Patch graft compositions for cell engraftment

ABSTRACT

Compositions and methods of transplanting cells by grafting strategies into solid organs (especially internal organs) are provided. These methods and compositions can be used to repair diseased organs or to establish models of disease states in experimental hosts. The method involves attachment onto the surface of a tissue or organ, a patch graft, a “bandaid-like” covering, containing epithelial cells with supporting early lineage stage mesenchymal cells. The cells are incorporated into soft gel-forming biomaterials prepared under serum-free, defined conditions comprised of nutrients, lipids, vitamins, and regulatory signals that collectively support stemness of the donor cells. The graft is covered with a biodegradable, biocompatible, bioresorbable backing used to affix the graft to the target site. The cells in the graft migrate into and throughout the tissue such that within a couple of weeks they are uniformly dispersed within the recipient (host) tissue. The mechanisms by which engraftment and integration of donor cells into the organ or tissue involve multiple membrane-associated and secreted forms of MMPs.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims priority under 35 U.S.C. § 119(e) to U.S.Application No. 62/518,380, filed Jun. 12, 2017, and to U.S. ApplicationNo. 62/664,694, filed Apr. 30, 2018, the contents of which are herebyincorporated by reference in their entirety.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Aug. 14, 2018, isnamed 069961-2804_SL.txt and is 6,662 bytes in size.

FIELD OF THE INVENTION

The present invention is directed generally to the field oftransplantation of cells or tissue engrafting. More specifically, fromsolid organs or tissues into solid organs or tissues, especially tointernal organs. The invention concerns compositions and methodsproviding strategies for the rapid transplantation, engraftment andintegration of cells into solid organs and tissues to treat diseases orconditions of solid organs or tissues, or to establish model systems ofa disease. Representative examples of this potential are cell therapiesfor treatment of hepatic or pancreatic diseases.

BACKGROUND OF THE INVENTION

There has long been a need for grafting strategies for cells from solidorgans, strategies distinct from those used for transplantation ofhemopoietic cells or for mesenchymal stem/progenitors. Turner, R., etal. Transplantation 90, 807-810 (2010); Gattinoni, L. et al. NatureMedicine 23, 18-27 (2017); Trounson A. et al. Cell Stem Cell 17, 11-22(2015); Sun B. K. et al. Science 346, 941-945 (2014); Lainas, P. et al.J Hepatol 49, 354-362 (2008). Transplantation of hematopoietic cells andof mesenchymal cells is done routinely by delivery of cells via avascular channel and is dependent on activation of adhesion molecules intransplanted cells when in relevant target sites because ofmicro-environmental signaling, a process referred to as “homing.”Methods used for skin (with similar ones for ocular targets) employgrafting methods with cells applied directly to target sites. Sun B. K.et al. Science 346, 941-945 (2014). Many grafting methods for skin areutilizable for cells from solid internal organs but require substantialmodifications to accommodate the microenvironment of these internalorgans. Grafts must contend with mechanical forces exerted byinteractions of tissues and organs on each other; examples include theeffects of lungs during breathing, or the compression of the liveragainst the diaphragm, or the transient effects of mechanical forcesexerted by the intestinal tract on neighboring tissues during processingof foods. Grafts, especially those for internal organs, are challengingto design because of concerns with respect to size, shape, andcomplexity in the structure of organs in addition to the dynamicmechanical forces evident.

For decades, cell therapies for cells from solid organs other than skinwere attempted using transplantation via a vascular route or by directinjection into the tissue. Most transplanted cells, when delivered byeither of these strategies, either die or are transported to ectopicsites, where they can live for months and create tissue in inappropriatesites, resulting potentially in adverse effects clinically. Turner, R.,et al. Transplantation 90, 807-810 (2010); Lanzoni, G. et al. Stem Cells31, 2047-2060 (2013). Engraftment in liver can be improved by coatingthe cells with hyaluronans and delivering them vascularly to the liver;the increased efficiency of engraftment is due to the liver's naturalprocess of clearance of hyaluronans. Nevi et al. Stem Cell Research &Therapy 8, 68, 2017. However, this improvement is still less efficientthan that with grafting strategies and, importantly, still allows fordelivery of cells to ectopic sites.

There remains a need for improved methods of cell engraftment into solidorgans. This disclosure fulfills this need and provides relatedadvantages.

SUMMARY OF THE INVENTION

There has long been a need for grafting strategies for cells from solidorgans (Turner, R., et al. Transplantation 90, 807-810 (2010),strategies distinct from those used for transplantation of hemopoieticcells, mesenchymal stem cells or for skin. Transplantation ofhemopoietic cells and mesenchymal cells is done routinely via a vascularchannel and is dependent on activation of adhesion molecules in relevanttarget sites because of micro-environmental signaling, a processreferred to as “homing”. Methods used for skin employ grafting methodswith cells applied directly to target sites.

Transplantation of cells from solid organs other than skin have longused vascular delivery. This is not logical, since adhesion molecules onthese cells are always activated and result in rapid (seconds) cellaggregation that can generate life-threatening emboli. Even if emboliare managed successfully to minimize health risks, the efficiency ofcell engraftment is low, only ˜20% for adult cells and even lower (<5%)for stem/progenitors. Most transplanted cells either die or aretransported to ectopic sites, where they can live for months, creatingtissue in inappropriate sites resulting in possible adverse effectsclinically. The small percentage of cells that engraft into target sitesintegrate slowly, requiring weeks to months to become a significantportion of the tissue. There is improvement in engraftment in liver ifcells are coated with hyaluronans and delivered vascularly due to thetissue's (e.g. liver's) clearance of hyaluronans. (Nevi et al. Stem CellResearch & Therapy 8, 68, 2017).

Applicants propose a radically different approach, one found even moresuccessful than coating cells with hyaluronans: placing grafts directlyonto the surface of the target site and using grafting biomaterials andthe unique phenotypic traits of certain cells when they are inconditions of the graft biomaterials to enhance transplantation. Thisparallels some aspects of strategies of cell therapies for skin butrequires substantial, modifications for internal organs given mechanicaleffects, abrasion or compression of organs near to each other, and giventhe unique fluid microenvironments around specific organs and the size,structure, and complexity of organs.

Described herein are novel patch graft compositions and methods fortransplantation of cells into tissue and solid organs. In someembodiments, the methods and grafts are adapted for internal organs,with design features dependent on the level of maturity of the cells,especially whether cells are stem cells or mature cells. In someembodiments, the donor cells (optionally autologous or allogenic) forthe patch grafts are disclosed herein incorporated into the graftbiomaterials in optionally as a mixture of cells or the form oforganoids, aggregates of epithelial stem cells and their native,lineage-stage appropriate mesenchymal cell partners—e.g. mesenchymalstem/progenitor cells such as early lineage stage mesenchymal cells(ELSMCs). In some embodiments, the donor cells are adult cellsincorporated into the graft materials as cell suspensions of adultepithelia and partnered with mesenchymal stem/progenitor cells,optionally ELSMCs, at ratios designed to optimize their expression ofmembrane-associated and/or secreted matrix metallo-proteinases (MMPs).In some embodiments, other variables of importance are the graftingbiomaterials and the backing material, both required to be neutral ineffects on the differentiation of the donor cells.

Aspects of the disclosure relate to a patch graft for sustaining andmaintaining a single cell population or a mixed population of cells,comprising: (a) a single cell type or a mixed population having two ormore cell types, at least one of which is at an early lineage stage thatis capable of expressing membrane-associated and/or secreted matrixmetalloproteinases (MMPs), or which has MMPs included from anothersource (e.g., purified or recombinant MMPs), said cell population ormixed population supported in a medium present in a hydrogel matrixhaving a viscoelasticity sufficient to allow for migration of said mixedpopulation, optionally, within or away from said hydrogel and/or withinor away from the patch graft; (b) a backing comprising a biocompatible,biodegradable material having a viscoelasticity sufficient to inhibit amigration of said mixed population in a direction of said backing; and,optionally, (c) a hydrogel overlaid on a serosal (i.e. outside) surfaceof said backing, which is opposite to that in contact with said mixedpopulation and, in embodiments where the patch graft is tethered to atarget site, is opposite the side in contact with the target site (e.g.organ or tissue). In some embodiments, this layer prevents or inhibitsadhesions by or from other tissues or organs. In some embodiments, thepatch graft is configured to sustain and maintain said mixed populationwhile inhibiting said at least one early lineage stage cell type fromdifferentiating or further maturing to a later lineage stage that is nolonger capable of expressing membrane-associated and/or secreted MMPs.The patch graft may be a single layer plus a backing or multiple layers.

In some embodiments, said backing is porous or non-porous. In someembodiments, the backing comprises a porous mesh, scaffold, or membrane.In some embodiments, the backing comprises silk; a synthetic textile; ora natural material such as aminion, placenta, or omentum; or acombination thereof. In some embodiments, said backing comprises aporous mesh infused with a hydrogel. In further embodiments, such aninfusion prevents cell migration away from the target organ or tissue.In some embodiments, said backing comprises a solid material.

In some embodiments, one or more of said hydrogels comprise hyaluronans.

In some embodiments, said medium comprises Kubota's medium or anothermedium supportive of stem cells and able to maintain stemness.

In some embodiments, said mixed population comprises mesenchymal cellsand epithelial cells. In some embodiments, said epithelial cells may beectodermal, endodermal, or mesodermal. In some embodiments, saidmesenchymal cells comprise early lineage stage mesenchymal cells(ELSMCs). In some embodiments, said ELSMCs comprise one or more ofangioblasts, precursors to endothelia, precursors to stellate cells, andmesenchymal stem cells (MSCs). In some embodiments, said epithelialcells comprise epithelial stem cells. In some embodiments, saidepithelial cells comprise biliary tree stem cells (BTSCs). In someembodiments, said epithelial cells comprise committed and/or matureepithelial cells. In some embodiments, said committed and/or matureepithelial cells comprise mature parenchymal cells. In some embodiments,said mature parenchymal cells comprise one or more of hepatocytes,cholangiocytes, and islet cells. In some embodiments, said mesenchymalcells and epithelial cells both comprise stem cells.

In some embodiment said mixed population comprises autologous and/orallogeneic cells.

In some embodiments, one or more cell types are genetically modified.

Further aspects related to methods employing the disclosed patch graftcompositions. Accordingly, provided herein are methods of engraftingcells into a target tissue comprising, consisting of, or consistingessentially of contacting the target tissue with a patch graft disclosedherein above.

In some embodiments of the methods, the target tissue is selected fromthe group consisting of liver, pancreas, biliary tree, thyroid, thymus,gastrointestine, lung, prostate, breast, brain, bladder, spinal cord,skin and underlying dermal tissues, uterine, kidney, muscle, bloodvessel, heart, cartilage, tendons, and bone tissue. In some embodimentsof the methods, the target tissue is liver tissue. In some embodimentsof the methods, the target tissue is pancreatic tissue. In someembodiments of the methods, the target tissue is biliary tree tissue. Insome embodiments of the methods, the target tissue is gastrointestinaltissue. In some embodiments, the tissue is diseased, damaged, or has adisorder. In some embodiments of the methods, the target tissue iskidney tissue.

In some embodiments of the methods, the target tissue is an organ. Insome embodiments of the methods, the organ is an organ of themusculoskeletal system, the digestive system, the respiratory system,the urinary system, the female reproductive system, the malereproductive system, the endocrine system, the circulatory system, thelymphatic system, the nervous system, or the integumentary system. Insome embodiments of the methods, the organ is selected from the groupconsisting of liver, pancreas, biliary tree, thyroid, thymus, stomach,intestines, lung, prostate, breast, brain, bladder, spinal cord, skinand underlying dermal tissues, uterus, kidney, muscle, blood vessel,heart, cartilage, tendon, and bone. In some embodiments, the organ isdiseased, damaged, or has a disorder.

Also provided herein are methods of treating a subject with a liverdisease or disorder, the methods comprising, consisting of, orconsisting essentially contacting the subject's liver a patch graftdisclosed herein above. In some embodiments of the methods, the liverdisease or disorder is liver fibrosis, liver cirrhosis, hemochromatosis,liver cancer, biliary atresia, nonalcoholic fatty liver disease,hepatitis, viral hepatitis, autoimmune hepatitis, fascioliasis,alcoholic liver disease, alpha 1-antitrypsin deficiency, glycogenstorage disease type II, transthyretin-related hereditary amyloidoisis,Gilbert's syndrome, primary biliary cirrhosis, primary sclerosingcholangitis, Budd-Chiari syndrome, liver trauma, or Wilson disease.

In other aspects, provided herein are methods of treating a subject witha disease or disorder of the pancreas, the methods comprising,consisting of, or consisting essentially of contacting the subject'spancreas with a patch graft disclosed herein above. In some embodimentsof the methods, the disease or disorder of the pancreas is diabetesmellitus, exocrine pancreatic insufficiency, pancreatitis, pancreaticcancer, sphincter of Oddi dysfunction, cystic fibrosis, pancreasdivisum, annular pancreas, pancreatic trauma, or hemosuccuspancreaticus.

In other aspects, provided herein are methods of treating a subject witha gastrointestinal disease or disorder, the method comprising,consisting of, or consisting essentially of contacting one or more ofthe subject's intestines with a patch graft disclosed herein above. Insome embodiments, the gastrointestinal disease or disorder isgastroenteritis, gastrointestinal cancer, ileitis, inflammatory boweldisease, Crohn's disease, ulcerative colitis, irritable bowel syndrome,peptic ulcer disease, celiac disease, fibrosis, angiodysplasia,Hirschsprung's disease, pseudomembranous colitis, or gastrointestinaltrauma.

In some aspects, provided herein are methods of treating a subject witha kidney disease or disorder, the methods comprising, consisting of, orconsisting essentially of contacting one or more of the subject'skidneys with a patch graft disclosed herein above. In some embodimentsof the methods, the kidney disease or disorder is nephritis, nephrosis,nephritic syndrome, nephrotic syndrome, chronic kidney disease, acutekidney injury, kidney trauma, cystic kidney disease, polycystic kidneydisease, glomerulonephritis, IgA nephropathy, lupus nephritis, kidneycancer, Alport syndrome, amyloidosis, Goodpasture syndrome, or Wegener'sgranulomatosis.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A-1D provides information about porcine donor cells for the patchgrafts. FIG. 1A is a schematic of the process and estimates of the timerequired for preparing organoids, assembling patch grafts and doing thesurgeries. In FIG. 1B, donor cells for the stem cell patch grafts wereisolated from cell suspensions of biliary tree tissue from transgenicpigs; the cells were prepared as organoids in serum-free Kubota's Mediumand on low attachment culture dishes. Organoids of biliary tree stemcells (BTSCs) and of their early lineage stage mesenchymal cell (ELSMCs)partners, angioblasts and precursors to endothelia and to stellatecells. They are shown in a phase micrograph versus one demonstratingexpression of the transgene, green fluorescent protein (GFP). All of thecells of the aggregate are green, since the transgene is in both theepithelial cells and the mesenchymal cells. The transgene was coupled tothe histone (H-2B) locus. Histology of the stem cell organoids that wereparaffin embedded, sectioned and stained with hematoxylin/eosin. (d)Magnified image of an organoid of BTSC and ELSMCs. FIG. 1C showsimmunohistochemistry (IHC) demonstrating expression of stem cell,hepatic and pancreatic markers indicating that these cells areprecursors to both liver and to pancreas. The IHC assays indicate outerlayers with intermediate stage stem cell markers such as EpCAM andinterior cells expressing very primitive genes such as pluripotencygenes and endodermal transcription factors (e.g. SOX17, SOX9, PDX1).FIG. 1D is a representative qRT-PCR assays assessing expression ofvarious genes in the organoids and indicating that cells are stem cellsor early progenitors. The controls were mature hepatocytes from pigletlivers.

FIGS. 2A-2F provides information about the major components of patchgrafts. FIG. 2A is a schematic of a patch graft affixed to the liver ofa pig, and on the right, the composition of the grafts. Early lineagestage cells, both the epithelia and the mesenchymal cells, are sourcesfor production of matrix metallo-proteinases (MMPs), key regulators ofengraftment. The matrix components of the graft biomaterials into whichdonor cells are placed are soft (˜100 Pa), without (or with minimal)sulfation, such as hyaluronan hydrogels. The structure of the graftconsist of layers of biomaterials and cells tethered to the target site.The medium components are devoid of serum, growth factors and cytokinesinfluential to differentiation of the donor cells and should be onestailored for survival and expansion of early lineage stage cells such asstem/progenitors. The backing has sufficient tensile strength to be usedin surgical procedures but be neutral in its effects on thedifferentiation of the donor cells (e.g. ones with type I collagenshould be avoided). The backing is impregnated or coated with a morerigid 10× hydrogel (˜700 Pa) to serve as a barrier to orient themigration of donor cells towards the target tissue and to minimizeadhesions. After attachment to the target site, a 2×HA hydrogel, onethat is sufficiently fluid to be coated or painted onto the serosalsurface, is added and used to further minimize adhesions. FIG. 2Bdepicts the graft affixed to the liver or the pancreas of a host. FIG.2C is a schematic of the graft demonstrating the layers constituting thegraft composition. FIG. 2D depicts the results of assays empiricallyassessing the rheological or viscoelastic properties (shear andcompressive mechanical forces) of the specific hydrogel layers. FIG. 2Eprovides a formulation of the viscoelastic properties of the 3 layers ofhydrogels. FIG. 2F is a close up image of a patch graft sutured to thesurface of the liver of a pig.

FIGS. 3A-3D depicts the result of immunohistochemistry (IHC) andhistology of the liver patch grafts. FIG. 3A (Panels (a)-(d)) shows theresults of Trichrome staining of the patch graft at one week. Trichromeidentifies collagens (blue), cytoplasm (red) and nuclei (black), and itwas used to identify Glisson's capsule (normally adjacent to the surfaceof liver lobules) and adhesions (on the serosal surface of the grafts).There is a high level of blue staining in the layers at the serosalsurface and implicate adhesions to the graft. Also, the graft hasseparated from the host tissue at the interface between the backing andthe host; this was found frequently due to the wealth of MMPs producedat this interface. The remodeling regions provide evidence of the lossof classic lobule structure of the liver; they result in a region inwhich the donor cells are migrating into the tissue and, in parallel,altering the host tissue structure. In low magnification images (a),Trichrome staining of grafts placed on to the liver validated thatextensive remodeling of the Glisson capsule was occurring and resultedoften in a separation between the graft and the host liver. In highermagnification images (b) the remodeling region is remarkably broad andconsisting of areas (c) near to the graft where liver lobule structureis missing altogether and (d) regions within the remaining liver lobulesthat are undergoing breakdown in the remodeling process. FIG. 3B (Panels(a)-(b)) shows the results of Trichrome staining of the patch graft atthree weeks. Hyaluronans in the graft have been resorbed leaving onlythe backing (a). With resorption of HA, the Glisson capsule reappears(b) and the liver lobules near to the graft have stabilized again intotheir typical histological patterns, such as lobule and acini for liver.The arrow in (b) indicates the reappearance of collagens in thereformation of the Glisson capsule. FIG. 3C (Panels (a)-(c)) and FIG. 3D(Panels (a)-(c)) shows the results of hematoxylin/eosin staining of asection from the grafts at one week post grafting (C) and two weeks postgrafting (D). The figures at the top are 40×. At sites within the figure(a,b,c) are enlargements that are magnified at 100×; the rectangularimage below each of these is magnified at 200×. Shown are 3 sites of thegraft: (a) a site within the backing and associated graft biomaterials;(b) a site at the interface between graft and host tissue; and (c) asite within the liver lobules. The hematoxylin/eosin staining yieldsimages that contribute to an appreciation of the engraftment andmigration process that incorporates features of inflammatory processes.

FIGS. 4A-4C shows engraftment, migration and rapid maturation to adultfates within a week. FIG. 4A is a low magnification image of the patchgraft on the surface of a pig liver after one week. The dashed lineindicates the interface of the graft and host liver. Donor GFP+ cells(with pink nuclei; white arrows indicate areas with large numbers of thedonor GFP+ stem cells) were visualized by labeling with an antibody toGFP and secondarily with one coupled to Novo Red, a red fluoroprobe.Nuclei were stained blue with 4,6-Diamidino-2-phenylindole (DAPI)enabling recognition of host cells having only blue nuclei and donorones having pink nuclei (merge of DAPI and the Novo Red). FIG. 4B(Sections (a)-(b)) show Host tissue (a) extending into the hyaluronans(HA, the black background) of the graft; tissue by the backing containsoccasional organoids (inset) but with most donor cells dispersed intosingle cells; large numbers of dispersed donor GFP+ stem cells (b) areseen throughout the host tissue. There is no evidence for the Glissoncapsule in this area that constitutes the region of remodeling. FIG. 4Cdemonstrates that engraftment and migration of donor cells was rapid;within a week, all donor cells were within the host liver; there weredonor cells both near the graft site and also on the opposite side ofthe liver lobe (estimate of the distance is at least 1.5 cm from thegraft). Ongoing studies are analyzing regions of the piglet livers atgreater distances (i.e. other lobes of the liver) to define moreprecisely how far the migration can go by the donor cells within adefined period of time. Shown are donor cells (pink nuclei) near lobulesof host mature hepatocytes (forest green color from auto-fluorescence oflipofuscins) on the distant side of the liver lobe from that of thegraft site. FIG. 4D (Panels (a)-(b)) shows that maturation of donorcells to adult fates occurred in parallel with HAs being resorbed.Enlargement of a region containing donor GFP+ cells (single cells withpink nuclei) near to host hepatocytes (a), forest green in color(autofluorescence of lipofuscins), and readily distinguished from maturedonor-derived (b) hepatocytes that are lavender in color (merge of thepink—GFP, blue—DAPI, and the green—lipofuscins), that is they werelineage restricted from donor GFP+ stem cells. With other IHC assays(data not shown), the bright, spring green color of cells amidst theplates of both host and donor hepatocytes proved to be endothelia andstellate cells.

FIGS. 5A-5C compares engraftment and maturation of cells in the liverpatch grafts after one week and two weeks post-transplantation. FIG. 5Ais an examination of porcine liver at 1 week after patch grafting.Sirius red stain, an azo dye staining collagens was used andimmunohistochemistry for pan-cytokeratin (pCK) and Sox9; andimmunofluorescence (IF) stains were performed on serial 3-μm sections.At the patch graft site, grafted donor cells merged with liver lobules.In the upper panels (original magnification=5×), patch grafts arecomposed of mesenchymal and epithelial pCK⁺ cells (arrows). In middlepanels, a higher magnification is provided (20×). Epithelial cells showan immunophenotype that is typical of biliary tree stem cells (BTSCs)expressing biliary cytokeratins (pCK) and the endodermal stem cellmarker Sox9. BTSCs within the patch graft are arranged in cell stringsreassembling bile ductules (arrows) and are in direct continuity withhepatocyte plates of the adjacent liver lobule (arrowheads). Hosthepatocytes in lobules are pCK and Sox9 negative. In lower panels(Original magnification=20×), the immunofluorescence for GFP allows oneto identify individual grafted cells and their progeny. Hepatocytes inlobules adjacent to the patch graft were GFP positive indicating thatthese were donor cells derived that had merged with host liverparenchyma. At the interface between patch graft and liver lobules,pCK⁺/GFP⁺ ductules (that is donor derived cholangiocytes) were in directcontinuity with GFP⁺/pCK⁻ cells (donor-derived hepatocytes) within thelobules (arrowheads) suggesting a maturation of grafting cells towardsan hepatocyte fate. FIG. 5B is an examination of porcine livers 2 weeksafter patch grafting. IF stains reveal that GFP⁺ cells are presentwithin lobules distant to the graft site. They are dispersed uniformlyand so are in a mix of host cells (ones with blue nuclei from DAPI) andof donor cells (pink/purple nuclei from merge of the blue from DAPI andthe red of the GFP label). They co-express mature hepatocyte markerssuch as Hepatocyte Nuclear Factor (HNF) 4α (a mix of green andpink/purple nuclei) and albumin (green cytoplasm and with pink/purplenuclei). Separate or merged channels were included. Nuclei weredisplayed in blue (DAPI). Original Magnification: 40×. FIG. 5C (Panels1-3) is an evaluation of porcine livers a week after patch grafting anddemonstrating the broad region of remodeling that occurs at theinterface between the patch graft and the host tissue. The section inthe low magnification image and in the enlarged image of 1 ishematoxylin/eosin (lightly stained); that in 2 is stained with Vector-SGproviding a blue/gray color; that in 3 is stained for alpha-fetoproteinwith hematoxylin/eosin background. Specific sites within 5C are numberedand correlate with enlargements that indicate the changes occurringwithin the lobules. The host liver lobules and acini are breaking downdue to the wealth of MMPs flooding into the area along with the donorcells. The donor cells are observed at the boundary regions of thelobules, sites also demonstrating liver-specific markers such as HNF4-aand α-fetoprotein, meaning that the cells are maturing to a liver fate.These traits were not expressed by the BTSCs and so these areindications that the donor cells are undergoing maturation to an hepaticfate.

FIGS. 6A-6D provides information about patch grafts of stem cellorganoids tethered to pancreas. FIG. 6A is a low magnification(panoramic scan) image of GFP+ donor cells that have engrafted into muchof the pancreas and into the submucosa of the duodenum (a regioncontaining Brunner's Glands). Immunofluorescent staining of pigpancreas, liver, and duodenum in the site of the patch graft. GFP(green), Insulin (red), DAPI (blue). Donor-derived GFP+ cells occur inthe proximity of the site where the patch graft was positioned, andappear integrated in the pancreas parenchyma. The silk mesh of the SERIsurgical scaffold is observed interposed among pancreas, liver, andduodenum. FIG. 6B shows that donor cells mature to functional islets. Athigher magnification, donor-derived GFP+/Insulin+ beta cells(yellow—from merge of the GFP and of the red from staining for insulin)are observed intermingled with host GFP−/Insulin+ (red) beta cells inthe pancreas parenchyma. Surrounding the islet cells are a large numberof GFP+ cells displaying a morphology consistent with that of pancreaticexocrine cells, including acinar and ductal cells. Supporting thisinterpretation are the findings in C and D that, indeed, these cells areproducing amylase, a classic acinar marker. FIG. 6C and FIG. 6D showevidence of functional acinar cells derived from donor stem cells.Immunofluorescent staining of a serial section from the same tissueblock in the site of the patch graft and with focus on the region ofengrafted GFP+ donor cells. Amylase (green), Insulin (red), Glucagon(white—not visible in the panoramic scan in C, but visible at the highermagnification in D), DAPI (blue). Amylase+ acinar cells are the vastmajority of the exocrine tissue of the pancreas. By comparing thestaining presented in the serial sections at low and highmagnifications, it is deduced that most of the donor-derived GFP+ cellsin the pancreas have acquired an amylase+ acinar fate.

FIGS. 7A-7H offers a characterization of matrix-metallo-proteinases(MMPs). MMPs are comprised of a large gene family of calcium-dependent,zinc-containing enzymes that dissolve extracellular matrix components.There are at least 24 isoforms known in pigs of which a subset aresecreted factors (e.g. MMP1, MMP2, MMP7, MMP9) and a subset aremembrane-associated (e.g. MMP14, MMP15). MMP1 was identified by IHC,especially in the areas of remodeling, but not by RNA-seq, since therehas not yet been an annotated form of porcine MMP1 available for RNA seqanalyses. FIG. 7A, FIG. 7B, FIG. 7C, and FIG. 7D show isoforms ofsecreted and membrane-associated categories were expressed by bothstem/progenitors and mature cells. Quantitation of the expression levelsindicated that the membrane-associated forms were similar for bothstem/progenitors and mature cells (note the comparisons in FIG. 7D). Bycontrast, secreted forms were expressed at very high levels instem/progenitors and at low or negligible levels in mature cell types.The cell populations of adult cells analyzed were isolated fromsuspensions of piglet livers and biliary tree tissue and comprised ofCD45+ cells (hemopoietic cells), CD146+ cells (stellate cells), CD31+cells (endothelia), EpCAM+/CD45− cells (adult diploid hepatocytes andcholangiocytes. These EpCAM+/CD45− cells are the mature parenchymalcells found in piglet livers. The BTSCs were isolated from the biliarytree by the protocols given in the examples. FIG. 7E showsrepresentative MMP expression in regions of remodeling with aBTSC/ELSMCs graft. In a section adjacent to the patch graft ofBTSCs/ELSMCs were stained with Trichrome indicating the region (bracket)of remodeling. The region appears as linear stripes of red and bluebeing cells and matrix components undergoing dissolution by the “sea” ofMMPs. The stripes end at the edges of lobules that are still mostlyintact but beginning to “fray” at their boundaries from the effects ofthe MMPs derived from the invading cells. FIG. 7F shows representativeimages of IHC assays for MMP1 (Novo-red+). Methyl green is thebackground stain. The liver's lobular/acinar structure has dissolvedinto the undulating swirls of cells and marked by the strong expressionof MMP1, a secreted isoform of MMPs. FIG. 7G shows a section stained forMMP2 (Novo-red+). Hematoxylin is the background stain. The liver'slobular/acinar structure has disappeared and has been replaced by a mixof cells with strong staining for MMP2 (rust brown color). FIG. 7H showsthe remodeling process ongoing within the liver lobules. The liverlobules have become strips of cells interspersed by invading cells;MMP2+ expression (rust colored) is very high and contributing to theloss of lobular/acinar structures. With clearance of hyaluronans (by 2-3weeks), the lobular structures reappeared.

FIG. 8 is a schematic demonstration of the engraftment and integrationphenomena in liver and on pancreas.

FIGS. 9A-9E provides information about patch grafts of mature (adult)hepatocytes partnered with mature mesenchymal cells (MMCs), such asendothelia or stellate cells. These patch grafts were unable to engraft.Engraftment was achievable if the hepatocytes were partnered with earlylineage stage mesenchymal cells (ELSMCs), here being porcine mesenchymalstem cells (MSCs). If presented with ELSMCs, then engraftment occurredbut with restriction to regions near to the graft. FIG. 9A (Panels(a)-(b)) shows Trichrome staining of normal pig liver. Bar is 200 μm forlow magnification image (a) and 50 μm for the higher magnification image(b). Note the collagens in the Glisson capsule and the boundariesbetween hepatic acini. FIG. 9B (Panels (a)-(b)) shows Trichrome stainingof patch graft of normal, adult hepatocytes partnered with maturemesenchymal cells (MMCs), endothelia and stellate cells, did notengraft. In the low magnification image (a) note that the Glissoncapsule is intact, and cells remain atop the capsule. (b) at the highermagnification, there is evidence of some remodeling (plasticityphenomena) of cells in the lobule next to the graft (the mottled redcolor within the hepatocytes). This plasticity is assumed due to themembrane-associated MMPs known to be present on both stem cells andadult cells. FIG. 9C (Panels (a)-(c)) shows IHC assays on patch graft ofnormal, adult hepatocytes partnered with mature mesenchymal cells(MMCs). At the higher magnification (a), it is evident that engraftmenthas not occurred. This section was stained with antibody to RBMY-1 andwith hematoxylin as the counterstain (b). The Glisson capsule is intactand so are the boundary zones between lobules, and (c) negative control(staining without primary antibody) to indicate non-specific staining.FIG. 9D (Panels (a)-(c)) shows Trichrome staining of patch graft ofnormal, adult hepatocytes partnered with ELSMCs that here were porcinemesenchymal stem cells (MSCs) played the role of a cellular source ofMMPs. The graft is separating at the interface between the graft and thehost tissue. The bracket indicates the region of remodeling. Note thatthe liver lobules have lost the matrix that normally constitutesboundary zones between them and appear frayed at the edges. In thehigher magnification (a) are seen donor cells (pale red compared withthe dark red ones in the centers of the lobules) throughout the image;in (b) is an enlargement of a region showing that the Glisson capsule isconsiderably thinner under the patch (compare with region to the left ofthe box) and in (c). Extensive remodeling was evident in the cellsadjacent to the graft (c). FIG. 9E (Panels (a)-(d)) shows a patch graftof hepatocytes partnered with ELSMCs (porcine MSCs) after one week. Thesection (a) was stained with antibody to RBMY-1 (brown) and with methylgreen as the counter stain. The donor cells engrafted (regions of rustred color) and matured into adult parenchymal cells in the acini near tothe graft. The section (b) shows an enlargement of the image near to theremains of the thinned Glisson capsule showed that donor cells (darkbrown nuclei) were interspersed uniformly with host cells (nuclei weremethyl green color). The section (c) is the negative control for (b).The section (d) was stained with antibody to GFP (coupled with Novus redand yielding a rust brown color) and with methyl green as the counterstain. Most of the cells have engrafted and formed a band of dark red,donor (mature) hepatocytes within the host liver acini. The Glissoncapsule remained but was diminished in thickness. Migration much beyondthe region of the liver near to the graft was not observed within thethree-week time-frame of the experiments.

FIG. 10 is a schematic comparing engraftment of stem cells versus adultcells.

FIG. 11 shows evidence that the engraftment process involves migrationof cells to considerable distances within the host tissue. Here isdemonstrated that for grafts of BTSCs/ELSMCs organoids at one weekpost-transplantation. The schematic of the liver divided into 8different zones is used to indicate the regions evaluated for thepresence of donor cells. Sections are prepared from the regions 1-8 andthen stained to enable identification of donor cells. In the table aresummarized the findings showing the distances between the graft and eachregion and the proportion of GFP+ cells found. The images to the left ofthe table are scans of a representative section from each zone. The darkbrown staining is strongest in 6 near to the graft and is fainter withincreasing distance from the graft, the palest being zone 1.

FIGS. 12A-12E provides evidence for migration of donor cells throughoutthe host liver. GFP+ cells stained with Novo-red (rust brown color);host cells are stained with methyl green. FIG. 12A (Panels (a)-(b)) is alow magnification image of interface of graft and the host liver. Theseparation of the graft from the host liver was often seen (note thisalso in FIG. 3) and was shown correlated with exceptionally high levelsof secreted MMPs. Enlargement of the regions (a) and (b) are givenbelow. Note the areas in the low magnification image and in theenlargement in (b) in which staining is mottled and with areas showing awashed out appearance and that proved due to hyaluronan levels in thetissue. FIG. 12B depicts the intermediate zones to which the cellsmigrated. Donor cells are throughout the tissue, both in bile ducts andin the parenchyma of the acini. FIG. 12C shows the distance zones towhich the cells migrated. Note that only the bile ducts are stained.FIG. 12D provides enlargements showing donor cells in bile ducts. FIG.12E provides enlargements within the parenchyma to show that the donorcells have GFP labeling in the nuclei.

FIGS. 13A-13D shows the adverse conditions obtained for patch graftswith certain backings (see also Tables 1 and 2). These includednecrosis, adhesions, and sites of cholestasis found to occur when graftswere placed too close to some ducts such that the swelling causedocclusion of the ducts.

FIG. 14 shows a chart of both lineage stages for epithelial cells (FIG.14A) and mesenchymal cells (FIG. 14B) and the corresponding biomarkerprofiles.

FIG. 15 (Panels A-E) shows organoids of H2B-GFP+ BTSCs/ELSMCs patchgrafted onto the Kidney. Evaluation was done at 1-week-post-grafting.Panel A shows Trichrome staining of grafted kidney. The kidney wasprepared in cross-section to expose the deeper layer that with the graftas a “V” shape. The lower half “V” with bright blue staining is thegraft side on the kidney; the upper “V” in the figure is a deeper layerto the grafted layer. Panel B shows H&E staining for the same section ofthe grafted kidney. Panel C is the higher magnification of the patchgrafted kidney. The capsule of the kidney under the graft was loosened(from dissolution by MMPs) in a fashion similar to that in the liver.Panel D shows IHC staining of GFP+ cells (dark red) that have engraftedinto the kidney at a layer under the patch. Panel E shows engraftment ofthe GFP+ cells (dark red) at deeper layers of the kidney. Necropsyreports indicated that there was no necrosis found in the grafted kidneyor elsewhere in the animals that were subjected to patch grafts.

BRIEF DESCRIPTION OF THE TABLES

TABLE 1 provides a summary of surgical or other approaches for patchgrafting.

TABLE 2 provides a comparison of backings tested for the exemplary patchgrafts.

TABLE 3 provides a summary of the antibodies used for IHC and IF in theexamples.

TABLE 4 provides a summary of the primers (SEQ ID NOS 1-28,respectively, in order of appearance) used for qRT-PCR assays.

DETAILED DESCRIPTION

Embodiments according to the present disclosure will be described morefully hereinafter. Aspects of the disclosure may, however, be embodiedin different forms and should not be construed as limited to theembodiments set forth herein. Rather, these embodiments are provided sothat this disclosure will be thorough and complete, and will fullyconvey the scope of the invention to those skilled in the art. Theterminology used in the description herein is for the purpose ofdescribing particular embodiments only and is not intended to belimiting of the invention. All publications, patent applications,patents and other references mentioned herein are incorporated byreference in their entirety.

Unless otherwise defined, all terms (including technical and scientificterms) used herein have the same meaning as commonly understood by oneof ordinary skill in the art to which this invention belongs. It will befurther understood that terms, such as those defined in commonly useddictionaries, should be interpreted as having a meaning that isconsistent with their meaning in the context of the present applicationand relevant art and should not be interpreted in an idealized or overlyformal sense unless expressly so defined herein. While not explicitlydefined below, such terms should be interpreted according to theircommon meaning.

The practice of the present technology will employ, unless otherwiseindicated, conventional techniques of tissue culture, immunology,molecular biology, microbiology, cell biology, and recombinant DNA,which are within the skill of the art. See, e.g., Sambrook and Russelleds. (2012) Molecular Cloning: A Laboratory Manual, 4rd edition; theseries Ausubel et al. eds. (2012) Current Protocols in MolecularBiology; the series Methods in Enzymology (Academic Press, Inc., N.Y.);MacPherson et al. (1991) PCR 1: A Practical Approach (IRL Press atOxford University Press); MacPherson et al. (1995) PCR 2: A PracticalApproach; Harlow and Lane eds. (2014) Antibodies, A Laboratory Manual,2d edition; Freshney (2011) Culture of Animal Cells: A Manual of BasicTechnique, 6th edition; Gait ed. (1984) Oligonucleotide Synthesis; U.S.Pat. No. 4,683,195; Hames and Higgins eds. (1985) Nucleic AcidHybridization; Anderson (1999) Nucleic Acid Hybridization; Hames andHiggins eds. (1984) Transcription and Translation; Immobilized Cells andEnzymes (IRL Press (1986)); Perbal (1984) A Practical Guide to MolecularCloning; Miller and Calos eds. (1987) Gene Transfer Vectors forMammalian Cells (Cold Spring Harbor Laboratory); Makrides ed. (2003)Gene Transfer and Expression in Mammalian Cells; Mayer and Walker eds.(1987) Immunochemical Methods in Cell and Molecular Biology (AcademicPress, London); and Herzenberg et al. eds (1996) Weir's Handbook ofExperimental Immunology.

Unless the context indicates otherwise, it is specifically intended thatthe various features of the invention described herein can be used inany combination. Moreover, the disclosure also contemplates that in someembodiments, any feature or combination of features set forth herein canbe excluded or omitted. To illustrate, if the specification states thata complex comprises components A, B and C, it is specifically intendedthat any of A, B or C, or a combination thereof, can be omitted anddisclaimed singularly or in any combination.

All numerical designations, e.g., pH, temperature, time, concentration,and molecular weight, including ranges, are approximations which arevaried (+) or (−) by increments of 1.0 or 0.1, as appropriate, oralternatively by a variation of +/−15%, or alternatively 10%, oralternatively 5%, or alternatively 2%. It is to be understood, althoughnot always explicitly stated, that all numerical designations arepreceded by the term “about.” It also is to be understood, although notalways explicitly stated, that the reagents described herein are merelyexemplary and that equivalents of such are known in the art.

Definitions

As used in the description of the invention and the appended claims, thesingular forms “a,” “an” and “the” are intended to include the pluralforms as well, unless the context clearly indicates otherwise.

The term “about,” as used herein when referring to a measurable valuesuch as an amount or concentration (e.g., the percentage of collagen inthe total proteins in the biomatrix scaffold) and the like, is meant toencompass variations of 20%, 10%, 5%, 1%, 0.5%, or even 0.1% of thespecified amount.

The terms or “acceptable,” “effective,” or “sufficient” when used todescribe the selection of any components, ranges, dose forms, etc.disclosed herein intend that said component, range, dose form, etc. issuitable for the disclosed purpose.

Also as used herein, “and/or” refers to and encompasses any and allpossible combinations of one or more of the associated listed items, aswell as the lack of combinations when interpreted in the alternative(“or”).

As used herein, the term “comprising” is intended to mean that thecompositions and methods include the recited elements, but do notexclude others. As used herein, the transitional phrase “consistingessentially of” (and grammatical variants) is to be interpreted asencompassing the recited materials or steps “and those that do notmaterially affect the basic and novel characteristic(s)” of the recitedembodiment. See, In re Herz, 537 F.2d 549, 551-52, 190 U.S.P.Q. 461, 463(CCPA 1976) (emphasis in the original); see also MPEP § 2111.03. Thus,the term “consisting essentially of” as used herein should not beinterpreted as equivalent to “comprising.” “Consisting of” shall meanexcluding more than trace elements of other ingredients and substantialmethod steps for administering the compositions disclosed herein.Aspects defined by each of these transition terms are within the scopeof the present disclosure.

As used herein, the term “patch graft” refers to a composition of cellsembedded or comprised in an appropriate biomaterial that allows fortransplanting donor cells (allogeneic or autologous) to the host. Insome embodiments, the term refers to a composition of cells embedded orcomprised in an appropriate biomaterial that allows for transplantingdonor cells to the host. Biomaterials are ones that can be preparedunder defined conditions (e.g., a basal medium optionally supplementedand/or a medium of nutritional factors, vitamins, amino acids,carbohydrates, minerals, insulin, transferrin/Fe, and/or lipids(purified free fatty acids complexed with purified albumin plus alipoprotein carrier molecule such as high density lipoprotein)) andcomprised, optionally solidified, into a soft gel (under 200 Pa,optionally approximately 100 Pa), and covered with a backing that hassufficient tensile strength to enable surgical attachment or otherwisetethered to a tissue or organ of the host and yet be of a chemistry withminimal effects on the differentiation of the donor cells. To be avoidedare supplements with factors that might drive differentiation of thecells, especially the early lineage stage mesenchymal cells (ELSMCs);these include serum, growth factors and cytokines affecting ELSMCs, andmature matrix components (e.g. type I collagen).

The term “backing,” as used herein, refers to a material that serves asa backing or barrier on the surface of the patch graft capable oftethering the graft to a target site and/or facilitating migration ofthe cells therein to the target site and/or preventing or inhibitingmigration of the cells toward the backing. The backing is or comprises a“biodegradable, biocompatible material,” “biocompatible, biodegradablematerial,” or any variation thereof referring to a material which (i) isbiocompatible with the subject into which it is being transplanted, (ii)exhibits mechanical resilience to withstand the compressive and shearforces that occur on organs and tissues (especially internal ones),which in turn enables this material to function as a surgical tissue,and (iii) has a neutral or minimal effect on the differentiation statusof cells that come in contact with the material. In some embodiments,the backing of the patch graft comprises such a material. In suchembodiments, the mechanical resilience of (ii) should be such that thebacking can be tethered the graft to the target site. In further suchembodiments, backing directs cell migration toward the target site—e.g.by affecting the differentiation of those cells migrating in directionsaway from the target site or by physically blocking said migration. Inthis regard, suitable materials include but are not limited toSeri-silk, optionally contour Seri-Silk, or derivatives thereof,aminions or extracts thereof (for example, of the side facing the fetusand/or a patch or textile comprised of PGA and/or PLLA. Non-limitingexamples of suitable patches of synthetic materials include a wovenpatch comprised of 91% PGA-co-9% PLLA, a knit patch comprised of 91%PGA-co-9% PLLA, or a non-woven patch comprised of 100% PGA. Moregenerally, suitable backings may include forms of Bombyx moth silk suchas Seri® Surgical Silk Scaffolds (Sofregen, New York, N.Y.), otherderivatives of Bombyx moth silk, and synthetic textiles, such as formsof Polyglycolic acid-co-poly-L-lactic acid (PGA/PLLA).

In some embodiments, the backing is also bioresorbable. As used herein,“bioresorbable” refers to a material that can be broken down by the bodyof a host or recipient of the graft and does not require mechanicalremoval. In some embodiments, the bioresorbable backing is bioresorbablewithin a span of about 2 to about 10 weeks, about 2 to about 20 weeks,about 2 to about 52 weeks, about 4 to about 16 weeks, about 4 to about12 weeks, or about 4 to about 8 weeks. In some embodiments, thebioresorbable backing is bioresorbable within a span of about 4 to about8 weeks; about 4 to about 12 weeks, about 4 to about 16 weeks, about 4to about 20 weeks, and about 4 to about 52 weeks.

As used herein, the biomaterials of the graft, and independent of thebacking, include ones that can form hydrogels. The term “gel” refers toa solid jelly-like material that can have properties ranging from softand weak to hard and tough. Gels are defined as a substantially dilutecross-linked system, which exhibits no flow when in the steady-state. Byweight, gels are mostly liquid, yet they behave like solids due to athree-dimensional cross-linked network within the liquid. It is thecrosslinking within the fluid that gives a gel its structure (hardness,stiffness, mechanical, or viscoelasticity properties) and contributes toits adhesivity. In this way gels are a dispersion of molecules of aliquid within a solid in which the solid is the continuous phase and theliquid is the discontinuous phase. A “hydrogel,” also referred to hereinas a “hydrogel matrix,” is a non-limiting example of a gel comprised ofa macromolecular polymer gel constructed of a network of polymer chains.Hydrogels are synthesized from hydrophilic monomers or hydrophilicdimers (e.g. in the case of hyaluronan) by either chain or step growth,along with network formation. A net-like structure along with voidimperfections enhance the hydrogel's ability to absorb large amounts ofwater via hydrogen bonding. As a result, hydrogels developcharacteristic firm yet elastic mechanical properties. They are able toundergo spontaneous formation of new bonds when old bonds are brokenwithin a material. The structure of the hydrogels along withelectrostatic attraction forces drive new bond formation throughnon-covalent hydrogen bonding.

The biomaterials used for the grafts have mechanical properties,stiffness, that can be more rigorously defined as the viscoelasticity ofthe biomaterials. See https://en.wikipedia.org/wiki/Viscoelasticity. Thegraft biomaterials conducive to engraftment must be very soft (forexample, about 100 Pa), conditions permissive for the donor cells toremain immature (Lozoya et al. Biomaterials 2011; 32 (30): 7389-7402.)and so be able to produce membrane-associated and/or secreted forms ofMMPs.

As used herein, the term “viscoelasticity” refers to the property ofmaterials that exhibit both viscous and elastic characteristics whenundergoing deformation. Viscous materials, like honey, resist shear flowand strain linearly with time when a stress is applied. Elasticmaterials strain when stretched and quickly return to their originalstate once the stress is removed. Viscoelastic materials have elementsof both of these properties and, as such, exhibit time-dependent strain.Whereas elasticity is usually the result of bond stretching alongcrystallographic planes in an ordered solid, viscosity is the result ofthe diffusion of atoms or molecules inside an amorphous material. Thoughthere are many instruments that test the mechanical and viscoelasticresponse of materials, broadband viscoelastic spectroscopy (BVS) andresonant ultrasound spectroscopy (RUS) are more commonly used to testviscoelastic behavior because they can be used above and below ambienttemperatures and are more specific to testing viscoelasticity. These twoinstruments employ a damping mechanism at various frequencies and timeranges with no appeal to time-temperature superposition. Using BVS andRUS to study the mechanical properties of materials is important tounderstanding how a material exhibiting viscoelasticity will perform

As used herein, the term “hyaluronan,” or “hyaluronic acid,” refers to apolymer of disaccharide units comprised of glucosamine and glucuronicacid [1-3] linked by β1-4, β1-3 bonds and salts thereof. Thus, the termhyaluronan refers to both natural and synthetic forms of hyaluronans.The naturally occurring hyaluronan (HA), water-soluble polysaccharidecomprising disaccharide units of D-glucuronic acid (GlcUA) andN-acetyl-D-glucosamine (GlcNAc), which are alternately linked, forming alinear polymer. High molecular weight HA may comprise 100 to 10,000disaccharide units. HAs often occur naturally as the sodium salt, sodiumhyaluronate. HA; sodium hyaluronate, and preparations of either HA orsodium hyaluronate are often referred to as “hyaluronan.” Non-limitingexamples of acceptable hyaluronate salts, include potassium hyaluronate,magnesium hyaluronate, and calcium hyaluronate.

Other glycosaminoglycans (GAGs) can also be used in the hydrogel. Theseinclude forms of chondroitin sulfate (CSs) and dermatan sulfates (DSs),polymers of glucuronic acid and galactosamine, and heparan sulfates(HSs) and heparins (HPs), polymers of glucuronic acid and glucosamine.The extent and pattern of sulfation of these GAGs are critical, sincethe sulfation patterns dictate the formation of complexes with multiplefamilies of proteins (e.g. coagulation proteins, growth factors,cytokines, neutrophilic enzymes). See, e.g., Powell A K, Yates E A,Fernig D G, Turnbull J E. Interactions of heparin/heparan sulfate withproteins: appraisal of structural factors and experimental approaches.Glycobiology. 2004 April; 14(4):17R-30R] Those appropriate for patchgrafts that optimize engraftment comprise hyaluronans, non-sulfatedGAGs, and ones with minimal sulfation such as forms of chondroitinsulfates found in stem cell niches, as shown in Karumbaiah L, et al.Chondroitin Sulfate Glycosaminoglycan Hydrogels Create Endogenous Nichesfor Neural Stem Cells. Bioconjug Chem. 2015 Dec. 16; 26(12):2336-49 andHayes A J, et al. Chondroitin sulfate sulfation motifs as putativebiomarkers for isolation of articular cartilage progenitor cells. JHistochem Cytochem. 2008 February; 56(2):125-38 (incorporated herein byreference).

As used herein, the term “cell” refers to one or more cells in thegraft. The cells of the present disclosure are eukaryotic. In someembodiments, this cell is of animal origin, optionally from a humanorgan, and can be a stem cell, a mature somatic cell, progenitor cell,or intermediates in the lineage stages from the stem cells to the maturecells. The term “population of cells” or “cells” refers to a group ofone or more cells of the same or different cell type with the same ordifferent origin; this term is used interchangeably herein with the term“donor cells,” which intend cells that may be autologous or allogeneic.In some embodiments, this population of cells may be derived from a cellline, from freshly isolated cells, or in some embodiments, thispopulation of cells may be derived from a portion of an organ or tissue,optionally from a donor or a recipient.

The term “stem cell” refers to cell populations that can self-replicate(produce daughter cells identical to the parent cell) and that aremultipotent, i.e. can give rise to more than one type of adult cell. Theterm “progenitor cell” or “precursor” as used herein, is broadly definedto encompass progeny of stem cells and their descendants. Progenitorsare cell populations that can be multipotent, bipotent, or unipotent buthave minimal (if any) ability to self-replicate. Committed progenitorsare ones that are unipotent and can differentiate into a particularlineage leading to only one mature cell type. Non-limiting examples ofstem cells include but are not limited to embryonic stem (ES) cells,induced pluripotent stem (iPS) cells, germ layer stem cells, determinedstem cells, (ectodermal, mesodermal or endodermal), perinatal stemcells, amniotic fluid-derived stem cells, mesenchymal stem cells (MSCs),angioblasts, and those derived from umbilical cord, Wharton's jelly,and/or placenta. Intermediates between stem cells and committedprogenitors include cell populations such as hepatoblasts and pancreaticductal progenitors and other forms of transit amplifying cells that maybe multipotent but have extensive proliferative potential but morelimited (if any) self-replicative ability.

The term “mesenchymal cells” refers to cells derived from themesenchyme, including but not limited to angioblasts, precursors toendothelia, precursors to stellate cells, endothelia, stellate cells,stromal cells, various subpopulations of mature and progenitor cells,and mesenchymal stem cells (MSCs) which are multipotent stromal cellsand various subpopulations of mature and progenitor mesenchymal cells.The MSCs are cell populations prepared by culture selection processesfrom tissues (Cathery et al. Stem Cells 2018; PMID:29732653; Graceb etal. Biochimie 2018: PMID 29698670; Caplan A I. Stem Cells Int. 2015;PMID: 26273305. There are at least two major categories of maturemesenchymal cells: (a) Mature mesenchymal cells (stellate/stromal cells)that produce and are surrounded by forms of extracellular matrix thatcomprise fibrillar collagens (e.g. type I, III, V) and associated matrixcomponents (fibronectins, chondroitin sulfate proteoglycans, dermatansulfate proteoglycans) and bound signals (e.g. growth factors,cytokines) that form a complex and bound signals (e.g. growthfactors/cytokines) that form a complex associated with cells that aretypically linear (string-like) cell populations. Nonlimiting examples ofsuch cells include stellate cells, tendon, stroma, and myofibroblasts.(b) Mature mesenchymal cells such as endothelia that produce and aresurrounded by forms of extracellular matrix that comprise networkcollagens (e.g. type IV, type VI, VIII, X) and associated matrixmolecules (laminins, heparan sulfate proteoglycans, heparinproteoglycans) and bound signals (e.g. growth factors, cytokines) thattogether are associated with cells having more squamous or cuboidal orcobblestone morphologies. Nonlimiting examples of such cells includeendothelia and myoepithelial.

The precursors to these mesenchymal cell types include but are notlimited to angioblasts which are multipotent and that can differentiateinto lineages of endothelia (the late stages of which are fenestratedendothelia) or stellate cells (the late stages of which aremyofibroblasts (stroma). The precursors also include mesenchymal stemcells (MSCs) which are multipotent cells and can differentiate intofibroblasts (stroma), osteoblasts (bone cells), chondrocytes (cartilagecells), myocytes (muscle cells) and adipocytes (fat cells)). The MSCsmay optionally be prepared by culture selection methods (Cathery et al.Stem Cells 2018; PMID:29732653; Graceb et al. Biochimie 2018: PMID29698670; Caplan A I. Stem Cells Int. 2015; PMID: 26273305.

The term “epithelial cell expansion” is correlated with the diameter ofa colony of epithelial cells that typically form colonies with cuboidalor cobblestone morphologies and with estimates of growth being thecomposite of the diameters of the cells of the colony. By contrast,estimates of growth of mesenchymal cell colonies are correlated with thedensity of the colony, since the mesenchymal cells are more migratoryand motile, and the colony density is a reflection of the net sum ofcells that remain within the colony boundaries.

The term “epithelial cells” refers to cells derived from the epithelium,specialized cells that provide diverse functions for the tissue and/orthe systemic needs of a host. They are recognized by their ability tomigrate as precursors or immature cells; with maturation, they becomestationary and form layers of squamous or cobblestone-like or columnarpolarized cells with apical, basal and lateral sides, and that are boundto each other by an assortment of junctions (connexins, tight junctions,adherens). Their expansion potential is indicated by the diameter of acolony (not by its density). The mature epithelial cells provide diversefunctions such as secretion of specialized products or contributions tometabolism (hepatocytes, cholangiocytes), detoxification (hepatocytes),production of enzymes (acinar cells), production of endocrine factors(e.g. islets or other endocrine cells)), electrical activity (neuronalcells), and absorption (intestinal cells).

The term “biliary tree stem cells” (BTSCs) refers to epithelial stemcells found throughout the biliary tree and located within peribiliaryglands (PBGs), Brunner's Glands, both extramural and intramural, as wellas within the crypts of gallbladder villi. They have the ability totransition into committed hepatic and/or pancreatic progenitor cells Thehepatic descendants enter into the liver sinusoids via canals of Hering;the pancreatic progenitors are found within pancreatic duct glands(PDGs), regions of the biliary tree located within the pancreas.

Thus far, at least 7 subpopulations of stem cell populations have beenidentified with overlapping traits and ranging from extremely primitiveBTSCs to stem cell populations definable as hepatic or pancreatic stemcells. Description of what is known for these is given below. The mostprimitive ones are found in both the extramural peribiliary glands—onestethered to the surface of the bile ducts—and; the intramuralperibiliary glands—ones found within the bile duct walls. The intramuralperibiliary glands (PBGs) near to the fibromuscular layer in the centersof the bile duct walls can also be considered crypts (with parallels tointestinal crypts), niches in which are found the most primitive stemcell populations. The largest numbers of the PBGs within the biliarytree network are found within the hepato-pancreatic common duct andwithin the large intrahepatic bile ducts. No PBGs occur in thegallbladder, and instead the stem cell niches within the gallbladder arethe bottoms of the gall bladder villi that contain intermediate to latestage stem cell populations that are precursors to hepatic stem cells.The BTSCs are precursors to both liver and to pancreas. They give riseto hepatic stem cells, precursors to liver, and to pancreatic stemcells, precursors to pancreas, and these are found throughout thebiliary tree but in numbers influenced by whether near to the liverversus the pancreas. Thus, small numbers of pancreatic stem cells andlarge numbers of hepatic stem cells are located in the PBGs of the largeintrahepatic bile ducts, whereas small numbers of hepatic stem cells andlarge numbers of pancreatic stem cells are located in the PBGs of thehepato-pancreatic common duct.

Summaries of genetic signatures are presented in the Figures. Ingeneral, all of the BTSCs subpopulations express generic biomarkers thatinclude endodermal transcription factors for both liver and pancreas(e.g. SOX9, SOX17, PDX1), pluripotency genes (e.g. OCT4, SOX2, NANOG,SALL4, KLF4/KLF5, BMI-1); one or more of the hyaluronan receptorisoforms (standard and/or variant isoforms) of CD44; CXCR4; andcytokeratins 8 and 18. Stem cell subpopulations within the biliary treeand PBGs include (1) Brunner's Glands stem cells in the submucosa of theduodenum and that express CK7, TRA-160 and 181 and with traitsdistinguishable from stem cells in the intestine; (2) early stageintramural Biliary Tree Stem Cell (BTSCs) that express sodium iodidesymporter (NIS) and CXCR4, OCT4, SOX2, NANOG, but do not express LGR5 orEpCAM; (3) intermediate stage intramural BTSCs that express less of NISbut gain expression of LGR5 but not EpCAM; (4) late stage intramuralBTSCs (the only BTSCs found in the gallbladder) and also found in highnumbers in the large intrahepatic bile ducts and in thehepato-pancreatic common duct. They express both LGR5 and EpCAM. Theseare precursors to hepatic stem cells (in the liver and expressing SOX17but not PDX1) and to the pancreatic stem cells (in the hepato-pancreaticcommon duct and expressing PDX1 but not SOX17); (5) hepatic stem cellsmay be found in the canals of Hering, in PBGs of the large intrahepaticbile ductules, in PBGs in the extrahepatic biliary tree; and in the PBGsof the hepato-pancreatic common duct, but the highest numbers are thoseat intrahepatic sites. The hepatic stem cells retain the ability toself-replicate and to be multipotent. The biomarkers for these cellsinclude SOX9, SOX17, HNF-4 alpha, ITGB1 (CD29), ONECUT 2, SALL4, LGR5,CD44, epithelial cell adhesion molecule (EpCAM) found in the cytoplasmand at the plasma membrane, neural cell adhesion molecule (NCAM), CD133(prominin), negligible levels (or none) of albumin, a complete absenceof alpha-fetoprotein (AFP), an absence of P450 A7, and an absence ofsecretin receptor (SR). Hepatic stem cells and hepatoblasts expresscytokeratins 8, 18 and 19; (6) pancreatic stem cells are found in smallnumbers throughout the biliary tree (even in the PBGs in the largeintrahepatic bile ducts) but are found in high numbers in PBGs of thehepato-pancreatic common duct. They have the pluripotency genes andexpression for the other genes noted for all of the stem cellpopulations, but they differ in no longer having SOX17; thesubpopulations that will lineage restrict to islets express NGN3. Theyexpress EpCAM throughout the cells and at the plasma membrane andexpress low (or no) insulin. Maturation of them is correlated withincreasing insulin expression as well as with expression of other islethormones (e.g. glucagon). Those maturing into acinar populations willexpress MUC6 and amylase.

It is noted that hepatic and pancreatic stem cells may also be found intheir respective source organs when they are early in development (e.g.as ESCs or otherwise), and that any of those cells disclosed herein maybe alternatively generated through induction (i.e. as iPSCs).

As used herein, the term “supportive” is used to describe cells whichare able to assist in the propagation of cells from another lineagestage or provide assistance to neighboring cells through the productionof “paracrine signals”, factors active in their effects on neighboringcells in terms of survival, expansion, migration, differentiation, andmaturation. For example, supportive mesenchymal cells may be defined bytheir ability to influence epithelial cells, optionally through thesecretion of matrix metallo-proteinases (MMPs) and/or one or moreparacrine signals or growth factors. Many of these are summarized inrecent reviews. (Cathery et al. Stem Cells 2018; PMID:29732653; Gracebet al. Biochimie 2018: PMID 29698670; Caplan A I. Stem Cells Int. 2015;PMID: 26273305.

The term “lineage stage partners” refers herein to mesenchymal cellsand/or epithelial cells that are lineage stage appropriate to supportengraftment of the cells. For the hepatic or biliary tree stem cells,these are comprised of angioblasts (CD117+, CD133+, VEGFr+,CD31-negative) and their immediate descendants, precursors to endothelia(CD133+, VEGFr+, CD31+, Van Wildebrand Factor (vWF+)) and precursors tostellate cells (CD146+, ICAM-1+, alpha-smooth muscle actin+(ASMA),vitamin A-negative). They can be mimicked, in part and/or to someextent, by use of mesenchymal stem cells (MSCs), such as but not limitedto ones derived from bone marrow or fat tissue. Not to be bound bytheory, it is believed that such cells should be used immediately afterisolation from tissue or after minimal passaging ideally underserum-free conditions. These cells are collectively referred to hereinas early lineage stage mesenchymal cells (ELSMCs).

Intermediates in the lineage network are referred to as “transitamplifying cells,” which are cells that can be bipotent (ormultipotent), have considerable proliferative potential but demonstratelittle (if any) true self-replication, have low to moderate (or even no)pluripotency gene expression, and express traits indicating commitmentto an hepatic (e.g. albumin, alpha-fetoprotein) or a pancreatic (e.g.insulin, MUC6, amylase) fate. These include hepatoblasts (the networkgiving rise to liver) and pancreatic ductal progenitors (the networkgiving rise to pancreas).

As used herein, the term “pancreatic ductal progenitors” refers tobipotent cells found within pancreatic ductal glands (PDGs) within thepancreas and giving rise to acinar cells and islets. In our studies, wefind that they express SOX9, PDX1, PTF1a, HNF1β, EpCAM, LGR5, ICAM-1,CD44, and subpopulations express NGN3 or MUC6 or amylase. They have beenextensively characterized by others. See, e.g., Rezanejad H,Ouziel-Yahalom L, Keyzer C A, Sullivan B A, Hollister-Lock J, Li W C,Guo L, Deng S, Lei J, Markmann J, Bonner-Weir S. Heterogeneity of SOX9and HNF1β is dynamic. Stem Cell Reports. 2018 Mar. 13; 10(3):725-738.

As used herein, the term “hepatoblasts” refers to bipotent hepatic cellsthat can give rise to hepatocytic and cholangiocytic lineages and arefound in or adjacent to canals of Hering or in PBGs within the largeintrahepatic bile ducts. They have an extraordinary ability toproliferate (that is expand) but with less ability (if any) toself-replicate relative to that observed in hepatic stem cells or BTSCs.These cells are characterized by a biomarker profile that overlaps with,but is distinct from, hepatic stem cells or biliary tree stem cells.They express SOX9, low (or even negligible) levels of SOX17, high levelsof LGR5, HNF4-alpha, and EpCAM, found primarily at the plasma membrane,and expressing P450A7, cytokeratin 7, secretin receptor, consistentexpression of albumin in all hepatoblasts, high levels ofalpha-fetoprotein (AFP), intercellular adhesion molecule (ICAM-1) but noexpression of NCAM, and negligible or no expression of pluripotencygenes (e.g. SALL4, KL4/KLF5, OCT4, SOX2, NANOG).) and no expression ofmature hepatic parenchymal markers (e.g. P450s such as P4503A).

As used herein the term “committed progenitor” refers to a unipotentprogenitor cell that gives rise to a single cell type, e.g. a committedhepatocytic progenitor cell. In some embodiments, they do not expresspluripotency genes. The committed hepatocytic progenitors are recognizedby expression of albumin, AFP, glycogen, ICAM-1, various enzymesinvolved with glycogen synthesis, and the gap junction gene, connexin28. These give rise to hepatocytes. A committed biliary (orcholangiocytic) progenitor gives rise to cholangiocytes and isrecognized by expression of EpCAM, cytokeratins 7 and 19, aquaporins,CFTR (Cystic Fibrosis Transmembrane Conductance Regulator), and membranepumps associated with production of bile. In some embodiments, acommitted islet progenitor expresses insulin, glucagon, and other islethormones albeit at low levels; with maturation the expression levels ofthe islet hormones increase but with particular cells expressingpreferentially certain hormones.

As used herein, the term “aggregates” refers to a plurality of cellsthat are amassed together. The aggregates may vary in both size andshape or may be substantially uniform in size and/or shape. The cellaggregates used herein can be of various shapes, such as, for example, asphere, a cylinder (preferably with equal height and diameter), orrod-like among others. Although other shaped aggregates may be used, inone embodiment of the disclosure, it is generally preferable that thecell aggregates be spherical or cylindrical. The term “non-aggregated”refers to individual, or single-celled, stem and/or progenitor cells ormature cells. In some embodiments, the compositions provided herein cancomprise substantially aggregated cells, substantially non-aggregatedcells, or a mixture thereof.

The term “organoid” refers herein to a particular cellular aggregate ofdonor epithelial cells with mesenchymal cells that is self-assembled bysimple panning methods described herein. In some embodiments, themesenchymal cells are supportive mesenchymal cells. In some embodiments,the organoids are formed after culturing on low attachment dishes andunder serum-free, defined conditions tailored to the lineage stage(s) ofthe aggregated cells in suspension. Others prepare organoids utilizingparticular matrix extracts, such as Matrigel. Indeed, this substance isknown to be the industry standard. See Hindley et al. Dev. Biology 2016;420:251-261. PMID:27364469. The conditions described in which theseorganoids are maintained will not work successfully for the use of theseorganoids in the patch grafts described in this invention. The factors,such as those found in Matrigel, will stop or substantially reduce theMMP production by the cells which is required for the success of thesepatch grafts. Moreover, Matrigel cannot be a components of conditionsfor cells to be used clinically in people or for veterinary purposes.

The term “culture” or “cell culture” means the maintenance of cells inan artificial, in vitro environment. A “cell culture system” is usedherein to refer to culture conditions in which a population of cells maybe grown ex vivo (outside of the body)

“Culture medium” is used herein to refer to a nutrient solution for theculturing, growth, or proliferation of cells. Culture medium may becharacterized by functional properties such as, but not limited to, theability to maintain cells in a particular state (e.g. a pluripotentstate, a proliferative state, quiescent state, etc.), to mature cells—insome instances, specifically, to promote the differentiation ofprogenitor cells into cells of a particular lineage. Non-limitingexamples of culture media are serum supplemented media (SSM) being anybasal medium supplemented with serum at levels that are typically about10% to about 20%. The serum can be autologous (the same species as thecells) or, more commonly, serum from animals that are routinelyslaughtered for commercial purposes (e.g. chickens, cows, pigs, etc.).Notably, the present embodiments involving stem cells employ media thatavoids incorporation of serum and/or serum components that drivedifferentiation. Kubota's medium, a serum-free medium designed forendodermal stem/progenitors and comprised of a basal medium medium(nutrients, amino acids, vitamins, salts, carbohydrates) with no copper,low calcium (<0.5 mM) and supplemented with selenium, zinc, insulin,transferrin, lipids but no cytokines or growth factors. Other mediafound supportive of stem cells might also be usable, but they must avoidany factors that cause the cells to differentiate, since thematurational process will result in muting of production ofmembrane-associate and/or secreted MMPs.

Basal media are buffers used for cell culture and are comprised of aminoacids, sugars, lipids, vitamins, minerals, salts, trace elements, andvarious nutrients in compositions that mimic the chemical constituentsof interstitial fluid around cells. In addition, cell culture media areusually comprised of basal media supplemented with a small percentage(typically 2-10%) serum. For the grafting technologies described herein,conditions are used to maintain the cells as stem cells or earlyprogenitor cells and so there is an avoidance of serum or any of thetypical supplements that might drive the cells towards a mature cellfate. In addition to the customary basal media, various nutritionalsupplements, lipids (mixture of free fatty acids complexed with albuminand carrier molecules such as high density lipoprotein). Only twohormone/growth factors are added: insulin needed for carbohydratemetabolism, and transferrin, needed as a Fe carrier for the polymerases.Kubota's medium, a serum-free medium designed for endodermalstem/progenitors is comprised of a basal medium (with no copped, lowcalcium (<0.5 mM) supplemented with zinc, selenium, insulin,transferrin, lipids but no cytokines or growth factors. Other growthfactors and cytokines and especially serum are to be avoided since theywill induce differentiation of the donor cells and, thereby, minimizethe production of MMPs, which are required for the engraftment andmigration processes.

“Kubota's Medium” as used herein refers to any medium containing nocopper, calcium (<0.5 mM), selenium, zinc, insulin, transferrin/Fe, amix of free fatty acids bound to purified albumin and, optionally, alsohigh density lipoprotein (HDL). In some embodiments, Kubota's Mediumcomprises any medium (e.g., RPMI 1640 or DMEM-F12) with no copper, lowcalcium (e.g., 0.3 mM), ˜10-9 M selenium, ˜0.1% bovine serum albumin orhuman serum albumin (highly purified and fatty acid free), ˜4.5 mMnicotinamide, ˜0.1 nM zinc sulfate heptahydrate, ˜10-8 M hydrocortisone(optional component used for hepatic but not pancreatic precursors), ˜5μg/ml transferrin/Fe, ˜5 μg/ml insulin, ˜10 μg/ml high densitylipoprotein, and a mixture of purified free fatty acids that are addedafter binding them to purified serum albumin. The free fatty acidmixture consists of ˜100 mM each of palmitic acid, palmitoleic acid,oleic acid, linoleic acid, linolenic acid, and stearic acid.Non-limiting, exemplary methods for the preparation of this media havebeen published elsewhere, e.g., Kubota H, Reid L M, Proc. Nat. Acad.Scien. (USA) 2000; 97:12132-12137, the disclosure of which isincorporated herein in its entirety by reference.

In some embodiments, the conditions of these patch grafts are,therefore, counter to the routine use of media supplemented with a smallpercentage (typically 2-10%) serum. Serum has long been added to providerequisite signaling molecules (hormones, growth factors, cytokines)needed to drive a biological process (e.g. proliferation,differentiation). In some embodiments, serum is not included to avoiddifferentiation of the cells and/or avoid inactivating or mutingproduction of MMPs, especially the secreted forms.

As used herein the term “amount effective” or “effective amount” refersto an amount that is sufficient to treat disease states or conditions(e.g. liver or pancreatic diseases). An effective amount can beadministered in one or more administrations, applications or dosages.Such delivery is dependent on a number of variables including the timeperiod during which the individual dosage unit is to be used, thebioavailability of the composition, the route of administration, etc. Itis understood, however, that specific amounts of the compositions forany particular patient depends upon a variety of factors including theactivity of the specific agent employed, the age, body weight, generalhealth, sex, and diet of the patient, the time of administration, therate of excretion, the composition combination, severity of theparticular disease (e.g. liver or pancreatic disease) being treated andform of administration.

The terms “equivalent” or “biological equivalent” are usedinterchangeably when referring to a particular molecule, biological, orcellular material and intend those having minimal homology while stillmaintaining desired structure or functionality.

As used herein, the term “expression” refers to the process by whichpolynucleotides are transcribed into mRNA and/or the process by whichthe transcribed mRNA is subsequently being translated into peptides,polypeptides, or proteins. If the polynucleotide is derived from genomicDNA, expression may include splicing of the mRNA in a eukaryotic cell.The expression level of a gene may be determined by measuring the amountof mRNA or protein in a cell or tissue sample; further, the expressionlevel of multiple genes can be determined to establish an expressionprofile for a particular sample.

As used herein, the term “functional” may be used to modify anymolecule, biological, or cellular material to intend that itaccomplishes a particular, specified effect.

The term “gene” as used herein is meant to broadly include any nucleicacid sequence transcribed into an RNA molecule, whether the RNA iscoding (e.g., mRNA) or non-coding (e.g., ncRNA).

As used herein, the term “generate” and its equivalents (e.g.generating, generated, etc.) are used interchangeable with “produce” andits equivalents when referring to the method steps that bring theorganoid of the instant disclosure into existence.

The term “isolated” as used herein refers to molecules or biologicals orcellular materials being substantially free from other materials.

The terms “nucleic acid,” “polynucleotide,” and “oligonucleotide” areused interchangeably and refer to a polymeric form of nucleotides of anylength, either deoxyribonucleotides or ribonucleotides or analogsthereof. Polynucleotides can have any three dimensional (3D) structureand may perform any function, known or unknown. The following arenon-limiting examples of polynucleotides: a gene or gene fragment (forexample, a probe, primer, EST or SAGE tag), exons, introns, messengerRNA (mRNA), transfer RNA, ribosomal RNA, RNAi, ribozymes, cDNA,recombinant polynucleotides, branched polynucleotides, plasmids,vectors, isolated DNA of any sequence, isolated RNA of any sequence,nucleic acid probes and primers.

A polynucleotide can comprise modified nucleotides, such as methylatednucleotides and nucleotide analogs. If present, modifications to thenucleotide structure can be imparted before or after assembly of thepolynucleotide. The sequence of nucleotides can be interrupted bynon-nucleotide components. A polynucleotide can be further modifiedafter polymerization, such as by conjugation with a labeling component.The term also refers to both double and single stranded molecules.Unless otherwise specified or required, any aspect of this technologythat is a polynucleotide encompasses both the double stranded form andeach of two complementary single stranded forms known or predicted tomake up the double stranded form.

The term “protein”, “peptide” and “polypeptide” are used interchangeablyand in their broadest sense to refer to a compound of two or moresubunit amino acids, amino acid analogs or peptidomimetics. The subunitsmay be linked by peptide bonds. In another aspect, the subunit may belinked by other bonds, e.g., ester, ether, etc. A protein or peptidemust contain at least two amino acids and no limitation is placed on themaximum number of amino acids which may comprise a protein's orpeptide's sequence. As used herein the term “amino acid” refers toeither natural and/or unnatural or synthetic amino acids, includingglycine and both the D and L optical isomers, amino acid analogs andpeptidomimetics.

As used herein, the term “subject” and “patient” are usedinterchangeably and are intended to mean any animal. In someembodiments, the subject may be a mammal. In some embodiments, themammal is bovine, equine, porcine, canine, feline, simian, murine,human, or rat. In some embodiments, the subject is a human.

The term “tissue” is used herein to refer to tissue of a living ordeceased organism or any tissue derived from or designed to mimic aliving or deceased organism. The tissue may be healthy, diseased,injured by trauma, damaged and/or have genetic mutations. The term“natural tissue” or “biological tissue” and variations thereof as usedherein refer to the biological tissue as it exists in its natural stateor in a state unmodified from when it was derived from an organism. A“micro-organ” refers to a segment of “bioengineered tissue” that mimics“natural tissue.”

The biological tissue may include any single tissue (e.g., a collectionof cells that may be interconnected) or a group of tissues making up anorgan or part or region of the body of an organism. The tissue maycomprise a homogeneous cellular material or it may be a compositestructure such as that found in regions of the body including the thoraxwhich for instance can include lung tissue, skeletal tissue, and/ormuscle tissue. Exemplary tissues include, but are not limited to thosederived from liver, pancreas, biliary tree, lung, intestines, thyroid,thymus thymus, bladder, kidneys, prostate, uterus, breast, skin andunderlying dermal tissues, brain, spinal cord, blood vessels (e.g.aorta, iliac vein,), heart, muscle, including any combination thereof.

As used herein, “treating” or “treatment” of a disease in a subjectrefers to (1) preventing the symptoms or disease from occurring in asubject that is predisposed or does not yet display symptoms of thedisease; (2) inhibiting the disease or arresting its development; or (3)ameliorating or causing regression of the disease or the symptoms of thedisease. As understood in the art, “treatment” is an approach forobtaining beneficial or desired results, including clinical results. Forthe purposes of the present technology, beneficial or desired resultscan include one or more, but are not limited to, alleviation oramelioration of one or more symptoms, diminishment of extent of acondition (including a disease), stabilized (i.e., not worsening) stateof a condition (including disease), delay or slowing of condition(including disease), progression, amelioration or palliation of thecondition (including disease), states and remission (whether partial ortotal), whether detectable or undetectable.

Abbreviations

AFP, α-fetoprotein; ALB, albumin; BTSCs, biliary tree stein cells; CD,common determinant; CD44, hyaluronan receptors; CD133, prominin; CFTR,cystic fibrosis transmembrane conductance regulator; CK, cytokeratinprotein; CXCR4, CXC-chemokine receptor 4 (also called fusin or C184;also called platelet factor 4; EGF, epidermal growth factor; ELSMCs,early lineage stage mesenchymal cells, consisting of angioblasts andtheir descendants, precursors to endothelia and to stellate cells;EpCAM, epithelial cell adhesion molecule; FGF, fibroblast growth factor;HBs, hepatoblasts; HGF, hepatocyte growth factor; HpSCs, hepatic stemcells; KM, Kubota's Medium, a serum-free medium designed for endodermalstem cells; KRT, cytokeratin gene; LGR5, Leucine-rich repeat-containingG-protein coupled receptor 5 that binds to R-spondin; MMPs, matrixmetallo-proteinases, a large family of proteinases associated withdissolution of extracellular matrix, with cell migration and withregenerative responses; NANOG, a transcription factor criticallyinvolved with self-renewal; NCAM, neural cell adhesion molecule; NIS,sodium/iodide symporter; OCT4, (octamer-binding transcription factor 4)also known s POU5F1 (POU domain, class 5, transcription factor 1), agene expressed by stem cells; PDX1, pancreatic and duodenal homeobox 1,a transcription factor critical for pancreatic development; PBGs,peribiliary glands, stem cell niches for biliary tree stem cells; SALL4.Sal-like protein 4 found to be important for self-replication of stemcells; SOX, Sry-related HMG box; SOX2, a transcription factor that isessential for maintaining self-renewal, or pluripotency in embryonic anddetermined stem cells. SOX9, transcription factor associated withendodermal tissues (liver, gut and pancreas; SOX17, a transcriptionfactor essential for differentiation of liver; VEGF, vascularendothelial cell growth factor; vWF, Von Willebrand Factor.

Modes of Practicing the Present Disclosure

In the examples provided herein, Applicants establish patch grafting, anovel method for transplantation of cells into internal organs withdesign features dependent on whether cells are stem cells or maturecells. Applicants demonstrate these methods herein with grafts ofbiliary tree stem cells (BTSCs), precursors to both liver and pancreas,and transplanted onto liver or pancreas. The hosts used for developingthese methods are breeds of swine, Sus scrofa domestics. They are majoranimal species used in translational research, surgical models, andprocedural training and are used increasingly as alternatives to monkeysin preclinical studies.

Exemplary success was achieved with organoids of biliary tree stem cells(BTSCs), precursors to liver and to pancreas, partnered with earlylineage stage mesenchymal cells (ELSMCs), and comprised in soft (˜100Pa) hyaluronan (HA) hydrogels. HA hydrogels, containing organoids, wereplaced onto Seri-silk backings (a mesh material) impregnated on theirserosal sides with more rigid HA hydrogel (˜700 Pa), and were surgicallyor otherwise tethered to the surface of the liver or pancreas. Within aweek, grafts caused remodeling of organ capsules and adjacent tissueand, optionally, distant parenchymal tissue followed by a merger ofdonor and host cells. By two weeks, donor cells had matured tofunctional adult fates such as hepatocytes (albumin) or islets(β-cells-insulin). By three weeks, with clearance of HAs, organ capsulesand normal tissue histology returned. Theengraftment/migration/integration processes proved dependent on multipleplasma membrane-associated and secreted matrix-metallo-proteinasesexpressed by the cells.

These results of these examples are in contrast to those from pastefforts to transplant cells from solid organs into internal organs, inwhich transplantation was accomplished either by direct injection or bydelivering cells via a vascular route (see reviews by Bhatia et al.,Lanzoni et al., Weber, and others). The past methods of transplantationresult in small numbers of cells being engrafted, in risks of embolithat can be life threatening, and in significant levels of ectopic celldistribution. These problems have caused cell therapies for internalsolid organs to be used minimally or not at all.

The patch graft strategy offers an alternative method for celltherapies, ones that can enable the delivery of adequate cell numbersand of their integration into the tissue to offer significantrestoration of function(s). The examples demonstrate safety so long asbiomaterials and the backing used were supportive of maintenance of someor all of the donor cells as immature and so able to produce therelevant repertoire of MMPs. A common source of failure was anyfactor(s) resulting in differentiation of the donor cells. Not to bebound by theory, it is contemplated herein that purified MMPs may beincorporated into graft biomaterials and/or cells may be transformed tosecrete MMPs using a recombinant expression system or other geneticmodification technique, as an alternative to providing a cells in thegraft which naturally produce the requisite MMPs. In such embodiments,the combination of MMPs incorporated or transduced via construct shouldinclude those identified in the expression profiles provided in theexamples below.

Composition of a Patch Graft

Aspects disclosed herein relate to a patch graft comprising a layercomprising a single population or two or more populations of cells (e.g.donor cells which may be autologous or allogeneic) and a source of MMPsand a backing comprising a biocompatible, biodegradable material, whichmay be used to tether the graft to a target site. In some embodiments,the population or populations of cells include a population ofepithelial cells and a population of mesenchymal cells. In someembodiments, the populations of cells must be maintained in a particularstate or “lineage stage” as part of the graft, meaning that they do notdifferentiate or mature further until incorporation into the organ. Thiscan be achieved by balancing variables relating to the cell source, MMPcontent, medium used, and backing qualities. Each of these aspects isdescribed in greater detail herein below.

Not to be bound by theory, it is believed that patch grafts can besuccessful with (1) an optimal cell population or mixture of cells—e.g.donor epithelial cells and a supporting mesenchymal stem/progenitor cellpopulation that generates membrane-associated and/or secreted MMPs—in amedium and hydrogel that does not lead to differentiation of thesupporting mesenchymal stem/progenitor cell population or that otherwisecontains appropriate MMPs, and (2) a backing suitable to tether thegraft to the target site and prevent migration of the cells in the grafttoward the backing, away from the target site.

Exemplary Cells

Not to be bound by theory, the cells may be at any maturational lineagestage including embryonic stem (ES) cells, induced pluripotent stem(iPS) cells, determined stem cells, committed progenitors, transitamplifying cells, or mature cells. However, in certain embodiments, asource of MMPs must be present in the patch graft. Thus, contemplatedherein are cellular sources of the MMPs for use in the patch grafts.Such cellular sources must be at an early lineage stage that is capableof expressing membrane-associated and/or secreted matrixmetalloproteinases. An non-limiting example of such an early lineagestage are early lineage stage mesenchymal stem cells (ESMLCs).

In some embodiments, the cells to be grafted are epithelial cellspartnered with mesenchymal cells. In some embodiments, the epithelialcells comprise epithelial stem cells. In some embodiments, theepithelial cells comprise committed and/or mature epithelial cells. Insome embodiments, the committed and/or mature epithelial cells comprisemature parenchymal cells. In some embodiments, the mature parenchymalcells comprise one or more of hepatocytes, cholangiocytes, or isletcells. In some embodiments, the mesenchymal cells comprise ELSMCs. Insome embodiments, the ELSMCs comprise one or more of angioblasts,precursors to endothelia, precursors to stellate cells, and MSCs. Insome embodiments, the epithelial cells and mesenchymal cells are lineagestage partners of one another. In some embodiments, the epithelial cellsand the mesenchymal cells are not lineage stage partners of one another,e.g. are not at approximately the same lineage stage or maturationstage, respectively. In some embodiments, the epithelial cells aremature cells. In some embodiments, the mesenchymal cells are ELSMCs.

In some embodiments, at least one of the epithelial cells and themesenchymal cells are derived from a donor. In some embodiments, thedonor is a subject in need of a tissue transplant. In some embodiments,the donor is the source of healthy cells for a tissue transplant. Insome embodiments, at least one of the epithelial cells and themesenchymal cells are autologous to an intended recipient of the patchgraft. In some embodiments, all of the cells (i.e. epithelial andmesenchymal) are autologous to the intended recipient of the graft. Insome embodiments, the donor of cells may be one other than the recipient(allograft) or may also be the subject (autologous) having the internalorgan in a diseased or dysfunctional condition, optionally, wherein areobtained from a portion of the internal organ that is not diseased ordysfunctional and/or that the cells have been genetically modified torestore function.

In another aspect, the mesenchymal cells are lineage-stage partners ofthe donor cells, e.g. at a comparable or corresponding lineage stage. Inanother aspect, the mesenchymal cells are not lineage-stage appropriatepartners of the donor cells. The mesenchymal lineage stage cells may beangioblasts, early lineage stage precursors to endothelia and/orstellate cells, mesenchymal stem cells, endothelia or stellate cells, orderivatives of these cell populations.

For stem cell transplants, epithelial cells should be partnered withtheir native, lineage stage partner mesenchymal cells (angioblastsand/or precursors to endothelia or to stellate cells). For adultepithelial cells, appropriate partners include early lineage stagemesenchymal cells (ELSMCs) that are comprised of angioblasts and/orprecursors to stellate cells and to endothelial cells. Applicants haveshown that one can use preparations of mesenchymal stem cells (MSCs) incombination with adult cells to achieve engraftment. In someembodiments, certain MSCs may be preferable to others. Not to be boundby theory, it is believed that grafts may be optimized by selectingcombinations of cells which require minimal, if any culturing of thecells and that will avoid serum and matrix components that might drivedifferentiation of the cells. Not to be bound by theory, it is furtherunderstood that the epithelial-mesenchymal relationship is important,since the paracrine signaling supports the production of MMPs. However,mature epithelial cells partnered with mature endothelia will survive inthe graft and will be functional cells but will not engraft. Thus, ifthe mature epithelial cells are partnered with mature stroma to form agraft, the resulting grafts are likely to become fibrotic.

For treatment of a diseased or dysfunctional organ, cells may be from adonor other than the recipient (allografts) or may also be autologoustransplants and so from the subject having the internal organ in adiseased or dysfunctional condition, optionally, wherein are obtainedfrom a portion of the internal organ that is not diseased ordysfunctional and/or that the cells have been genetically modified torestore function.

For establishing a model system to study a disease, cells can be onesthat have the disease and that are transplanted onto/into normal tissuein an experimental host.

In some embodiments, the epithelial cells may be stem cells combinedwith supportive mesenchymal cells, optionally ELSMCs, to form organoids,which optionally self-assemble. These organoids may be embedded orcomprised in a hyaluronan hydrogel. The stem and/or progenitor cells ofthe present disclosure can include any stem and/or progenitor cell knownin the art, including for example, an embryonic stem cell (ESC), anembryonic germ cell (EGC), an induced pluripotent stem cell (iPSC), apancreatic stem cell (PSC), hepatic stem cell (HpSC), biliary tree stemcell (BTSC), an hepatoblast, a pancreatic ductal progenitor, a committedpancreatic progenitor cell, or a committed hepatic progenitor cell. Insome embodiments, the cell populations comprise only stem cells such aspancreatic stem cells, hepatic stem cells, biliary tree stem cells(BTSCs) or Brunner's Glands stem cells. In other embodiments, the cellscomprise only multipotent progenitor subpopulations such as hepatoblastsor pancreatic ductal progenitor cells, or the graft can containcommitted, unipotent progenitors (e.g. hepatocytic or biliary or isletor acinar committed progenitor cells). In other embodiments, the cellscomprise a mixture of stem cells and progenitors.

If adult epithelial cells are used, then they may be mixed at relevantratios with ELSMCs into the grafting biomaterials. The ratios of cellmixture may be determined so as to mimic the target tissue.Alternatively or in addition, the ratios may be determined throughself-assembly of the organoids. The organoids or cell mixtures areembedded in the soft grafting biomaterials such as the soft hyaluronanhydrogel. If a stem cell graft, then the stem and/or progenitor cells ofthe present disclosure can include any stem and/or progenitor cell knownin the art, including for example, an embryonic stem cell (ESC), anembryonic germ cell (EGC), an induced pluripotent stem cell (iPSC), aBrunner's Glands stem cells (BGSCs), a biliary tree stem cell (BTSC), apancreatic stem cell (PSC), an hepatic stem cell (HpSC), transitamplifying cells (e.g. hepatoblasts or pancreatic ductal progenitors),and committed, unipotent progenitors (e.g. a committed pancreaticprogenitors or hepatocytic or cholangiocytic progenitor). In someembodiments, the cell populations comprise only stem cells. In otherembodiments, the cells comprise only progenitor subpopulations. In otherembodiments, the cells comprise a mixture of stem cells and progenitorsor a mixture of stem/progenitor cells and more mature cells. In yetothers, there can be a chimeric mix of adult cells (e.g. hepatocytes,cholangiocytes, enterocytes, islets) and ELSMCs.

The stem cell and/or progenitor cells can be identified by any methodknown to one who is skilled in the art. Non-limiting examples includeusing a combination of assays defining self-replicative ability and onesdemonstrating multipotency by morphological analysis, by gene and/orprotein expression, cell surface markers, and the like. In someembodiments, the stem and/or progenitor cells express at least onemarker indicative of early stage liver cell lineage cell (e.g., SOX 17,HNF-4alpha, HNF6, HES1, CK19),) and at least one marker indicative ofearly stage pancreatic cell lineage (e.g., PDX1, PROX1, NGN3, HNFβ1).For example, stem and/or progenitor cells, in particular BTSCs, can beidentified by expression of SOX9, SOX17, PDX1, CD133, NCAM, sonichedgehog (SHH), sodium iodide symporter (NIS), LGR5, LGR6, EpCAM,various isoforms of CD44, CXCR4, and various pluripotency genes (e.g.OCT4, SOX2, NANOG, KLF4, KLFS, SALL4, BMi-1) or any combination thereof.

In some embodiments, the stem and/or progenitor cells express at leastone marker indicative of early parental stage cell lineages such asparental lineages for liver and pancreas. Thus they would express one(s)shared by both hepatic and pancreatic lineages (e.g. SOX9, LGR5/LGR6,EpCAM, CD133, CK19) and one(s) for hepatic lineages (e.g., SOX 17,HNF-4-alpha, HNF6, HES1) and one(s) for early stage pancreatic celllineages (e.g., PDX1, PROX1, NGN3, HNFβ1). For example, stem and/orprogenitor cells, in particular BTSCs, can be identified by expressionof SOX9, SOX17, PDX1, CD133, NCAM, sonic hedgehog (SHH), sodium iodidesymporter (NIS), LGR5, LGR6, EpCAM, and various pluripotency genes (e.g.OCT4, SOX2, NANOG, KLF4, KLF5, SALL4, BMi-1) or any combination thereof.

Generation of Mature Cell Types

The stem and/or progenitor cells can also be differentiated into a moremature cell type, if one is desired. This can be done in vitro byspontaneous differentiation and/or by directed differentiation. Directeddifferentiation can involve use of a defined media, geneticallymodifying the stem and/or progenitor cells to express a gene ofinterest, or combinations thereof.

Non-limiting examples of defined media to differentiate cells includethe hormonally-defined media (HDM) used for differentiation ofendodermal stem cells to adult fates. Supplements can be added toKubota's Medium to generate a serum-free, hormonally defined medium(HDM) that will facilitate differentiation of the normal hepatic orbiliary tree stem cells to specific adult fates. These includesupplementation with calcium to achieve at or above 0.6 mMconcentration, 1 nM tri-iodothyronine (T3), 10⁻¹²M copper, 10 nM ofhydrocortisone and 20 ng/ml of basic fibroblast growth factor (bFGF).The medium conditions over and above these needed to selectively yieldhepatocytes (HDM-H) versus cholangiocytes (HDM-C) versus pancreaticislets (HDM-P) are:

-   -   1) HDM-H: supplementation further with 7 μg/L glucagon, 2 g/L        galactose, 10 ng/ml epidermal growth factor (EGF) and 20 ng/ml        hepatocyte growth factor (HGF);    -   2) HDM-C: supplementation further with 20 ng/ml vascular        endothelial cell growth factor (VEGF) and 10 ng/ml HGF; and    -   3) HDM-P: prepared without glucocorticoids and further        supplemented with 1% B27, 0.1 mM ascorbic acid, 0.25 μM        cyclopamine, 1 μM retinoic acid, 20 ng/ml of FGF-7 for 4 days,        then changed with one supplemented with 50 ng/ml exendin-4 and        20 ng/ml of HGF for 6 more days of induction.

The HDM provided herein can be supplemented with additional growthfactors including, but not limited to, Wnt signals, epidermal growthfactors (EGFs), fibroblast growth factors (FGFs), hepatocyte growthfactors (HGFs), insulin-like growth factors (IGFs), transforming growthfactors (TGFs), nerve growth factors (NGFs), neurotrophic factors,various interleukins, leukemia inhibitory factors (LIFs), vascularendothelial cell growth factors (VEGFs), platelet-derived growth factors(PDGFs), stem cell factors (SCFs), colony stimulating factors (CSFs),GM-CSFs, erythropoietin, thrombopoietin, heparin binding growth factors,IGF binding proteins, and/or to placental growth factors.

The HDM provided herein can be supplemented with cytokines including,but not limited to interleukins, lymphokines, monokines, colonystimulating factors, chemokines, interferons and tumor necrosis factor(TNF).

Applicants have shown that hyaluronans can influence stem and/orprogenitor cells to express factors that regulate critical cell adhesionmolecules needed for cell attachment and cell-cell interactions and toprevent the stem and/or progenitor cells from internalization of thoseattachment factors following cell suspension preparations,cryopreservation, or with transplantation. Non-limiting examples of suchattachment factors include integrins. Integrins are a large family ofheterodimeric transmembrane glycoproteins that function to attach cellsto extracellular matrix proteins of the basement membrane, ligands onother cells, and soluble ligands. Integrins contain a large and smallsubunit, referred to as α and β, respectively. This subunits form aβheterodimers and at least 18 α and eight β subunits are known in humans,generating 24 heterodimers. In some embodiments, the stem and/orprogenitor cells express higher levels of integrin subunits, forexample, ITGα1, ITGα2, ITGα2B, ITGα3, ITGα4, ITGα5, ITGα6, ITGα7, ITGα8,ITGα9, ITGα10, ITGα11, ITGαD, ITGαE, ITGαL, ITGαM, ITGαV, ITGαX ITGβ1,ITGβ2, ITGβ3, ITGβ4, ITGβ5, ITGβ6, ITGβ7 and ITGβ8. In one preferredembodiment, the stem and/or progenitor cells express higher levels ofintegrin subunit beta 1 (ITGβ1) and/or integrin subunit beta 4 (ITGβ4).Takada Y. et al. (2007) Genome Biol. 8(5): 215.

In some embodiments, the stem and/or progenitor cells of the presentdisclosure differ from naturally occurring stem and/or progenitor cellsat least in that they express an integrin subunit in an amount that isat least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%,70%, 75%, 80%, 85%, 90%, 95%, 100%, 200% greater than the amount of theintegrin subunit in unmodified stem and/or progenitor cells. It iscontemplated that an increase in an integrin subunit can help the stemand/or progenitor cell to attach, form cell-cell interactions, and toprevent the stem and/or progenitor cells from internalization shouldthis be desired.

MMPs

The MMPs are one of the key factors facilitating engraftment andintegration. MMPs are comprised of many isoforms (at least 28; in thepigs, 24 isoforms are known) of which some are secreted (e.g. MMP1,MMP2, MMP7, MMP9) and some are plasma membrane associated (e.g. MMP14,MMP15). Not to be bound by theory, it is believed that a mix of these isrequired for engraftment, especially a mix of the secreted forms. Allcells examined produce varying amounts of both secreted and membraneassociated forms, but stem/progenitors produce very high levels of thesecreted forms. Engraftment is dependent on these secreted MMPs (andwith some known synergies with the membrane-associated forms). Acellular source of these is the practical way to provide the requisiteMMPs to achieve engraftment. As an alternative approach, Applicantscontemplate incorporation of purified/recombinant forms of the MMPs intothe graft biomaterials and/or genetic engineering of cells in the graftto produce the requisite MMPs.

The cells can successfully engraft as long as there are sources, ideallycellular sources, of multiple matrix metallo-proteinases (MMPs),optionally one or both of secreted and membrane-associated ones. MMPsare produced by all cell types, both immature and mature cells, but theyvary as to which isoforms are produced and at what level of expressionof particular MMPs. Representative secreted ones include MMP1, MMP2,MMP7 and MMP9. Representative membrane-associated ones include MMP14 andMMP15. Empirically it has been found that the highest production ofsecreted MMPs is by early lineage stage cells, stem cells and earlyprogenitors. The biomaterials of the graft support the ability of boththe epithelial and mesenchymal cells to produce these multiple forms ofmatrix metallo-proteinases (MMPs) that dissolve capsules around organsor tissues and enable migration of cells by means of dissolution ofmultiple forms of extracellular matrix components.

More generally, matrix metallo-proteinases (MMPS) are a large family ofzinc-dependent proteinases that are involved in breakdown and modulationof extracellular matrix component and that are involved in implantation,invasion, angiogenesis, vascularization, and migration in normal andpathogenic processes. There are at least 28 isoforms that comprisematrixins, adamalysins, astacins, serralysins, etc. Their roles havebeen characterized in normal processes such as the implantation of theplacenta, as well as in pathogenic ones such as invasion and metastasesof cancers.

The studies described herein offer evidence for entirely new roles ofMMPs that contribute to engraftment, migration and integration oftransplanted cells. Stem/progenitors, both epithelial ones andmesenchymal ones, express multiple MMP isoforms that are especiallypotent in these roles. Maturation of the cells results in muting theexpression of one or more of the potent stem/progenitor-cell-associatedMMPs and so diminishing the invasion and migration processes. Adultcells also express MMPs, primarily ones that are membrane bound(MT-MMPs), said MMPs are involved in plasticity processes but not thewholesale engraftment and integration of cells into tissues. However,there are some synergies between the MT-MMPs and the secreted forms. Thenet sum of this realization is that the graft biomaterials, backing andother conditions must be ones that, among other characteristics,optimize expression of the various MMPs, such as the secreted MMPs,enabling the grafting and migration processes to occur. Therefore,factors driving differentiation of the transplanted cells will, inparallel, mute the complex MMP responses. This realization means thatfactors to be avoided include serum (which drives differentiation),soluble signals that drive differentiation (e.g. certain growth factors,cytokines and hormones); extracellular matrix components that drivedifferentiation (e.g. collagens, adhesion molecules, highly sulfatedglycosaminoglycans/proteoglycans); and mechanical forces contribute torigidity (the viscoelasticity properties, which drive differentiation)of the graft.

In some embodiments, one or more of the cells in the mixture is a sourceof secreted and/or membrane-associated MMPs. The secreted MMPs mayoptionally be produced naturally by the one or more of the epithelial ormesenchymal cells or optionally be produced due to transformation of theone or more of the epithelial or mesenchymal cells with a recombinantexpression vector or genetic editing for MMP production. In someembodiments, such as but not limited to those involving stem/progenitorcell populations that naturally secrete MMPs, variables that mute MMPexpression—optionally membrane-associated and/or secreted MMPexpression—are controlled in the patch graft. Non-limiting examples ofsuch variables include variables that result in maturation ofstem/progenitor cells, such as but not limited to serum supplementationto media or to the graft biomaterials, hormones or other soluble signalsthat influence differentiation of the epithelial and/or mesenchymalcells, oxygen levels (as anaerobic conditions keep the cells immature,whereas higher oxygen levels promote differentiation), and the rigidityof graft materials (as rigidity or mechanical forces such as shear forceand compression may drive differentiation).

For stem cell grafts, both the epithelial cells and their mesenchymalcell partners are optimally stem cells or progenitors, since bothprovide contributions of multiple types of MMPs. To engraft adult cells,the one of the epithelial or mesenchymal cells should optimally providea cellular source of membrane-associated and/or secreted MMPs, e.g.optionally using ELSMCs as the cellular source of membrane-associatedand/or secreted MMPs. Thus, grafts in which both the epithelia and themesenchymal cells are mature cell types are not successful forengraftment. If mature endothelia, then the epithelial cells are likelyto survive and to proliferate and function but will not engraft; ifmature stroma, then the grafts are likely to become fibrotic.

In summary, engraftment will occur if both epithelial-mesenchymal cellpartners are stem/progenitors or if there at least one of the epithelialor mesenchymal cells is a stem cells, e.g. optionally using ELSMCs as asource of matrix-associated and/or secreted isoforms of matrixmetalloproteases (MMPs), or if purified/recombinant forms of those MMPsare provided in the graft biomaterials. The early lineage stagemesenchymal cells (ELSMCs) appropriate for patch grafts can beangioblasts, precursors to endothelia, early lineage stage endothelia,precursors to stellate cells, early stage stellate cells, or mesenchymalstem cells (MSCs), or mixtures of these.

Thus, contemplated herein is a composition for use as a patch graftcomprising at least a population of cells (e.g. epithelial andmesenchymal cells) and a source of MMPs (i.e. a population of cells atan early lineage stage that is capable of expressing membrane-associatedand/or secreted matrix metalloproteinases (MMPs), optionally supportedby the conditions of the medium and/or hydrogel.

Medium Components

For use in combination with the cells and source of MMPs disclosedherein, one can use any medium (comprising nutrients, vitamins, salts,etc.) plus critical soluble factors such as insulin, transferrin/Fe andlipids that is found useful for expansion and/or survival ofstem/progenitors. One must avoid all factors that cause the cells tomature, since maturation will result in a reduction or muting ofexpression of MMPs. The factors to be avoided include serum, solublesignals that drive differentiation, extracellular matrix components thatdrive differentiation, and rigidity or mechanical forces (compression,abrasion). A non-limiting example of such a media is Kubota's medium.

Thus, contemplated herein is a composition for use as a patch graftcomprising at least a population of cells and a source of MMPs (e.g. apopulation of cells at an early lineage stage that is capable ofexpressing membrane-associated and/or secreted matrix metalloproteinases(MMPs), supported in a suitable medium, or purified MMPs). Anon-limiting example of a suitable medium is Kubota's medium. Other stemcell mediums, such as those used for embryonic stem (ES) cells orinduced pluripotent stem (iPS) cells may likewise be suitable as long asthey do not contain soluble signals or matrix signals that will drivethe differentiation of the cells that are the source of the MMPs or aslong as MMPs are present or included from other sources.

Hydrogel

The patch graft comprises one or more hydrogel components. In someaspects, the biomaterials that can form hydrogels, or a parallelinsoluble complex (e.g. a non-collagenous gelatin), comprisehyaluronans, thiol-modified hyaluronans, other glycosaminoglycans(GAGs), or combinations thereof. A trigger for solidification can be anyfactor eliciting cross-linking of the matrix components or gelation ofthose components that can gel. The cross-linker may comprisePoly(ethylene glycol) (PEG) or PEG-diacrylate (PEGDA) hydrogel or adisulfide-containing derivative thereof. Notably, biomaterials comprisedin the hydrogel should be selected for the ability to support thestemness in the one or more cell populations disclosed for use in thepatch graft, e.g. ELSMCs.

Matrix components supportive of maintenance of stemness can be used butnot those components driving differentiation. Non-limiting examples ofsupportive components include hyaluronans or non-sulfated (ormiminally-sulfated) glycosaminoglycans. These are especially usefulsince thy can be “tuned,” that is modified to having varying levels ofrigidity (optionally measured as viscoelasticity). Accordingly, in someaspects, the population of cells, optionally isolated cells of aninternal organ, may be solidified ex vivo within the biomaterials priorto introducing the cells into the hosts, or in the alternative, injectedas a fluid substance and allowed to solidify in vivo.

The very soft versions (e.g. ˜100 Pa) of hydrogels are ideal formaintaining the donor cells in an immature state). More rigid versions(e.g. >500 Pa) can be used to cause the cells to mature enough to shutoff MMP production and so block migration. More rigid versions can alsominimize adhesions from neighboring tissues. In certain embodiments, thepopulation of cells and the source of MMPs, optionally anotherpopulation of cells (i.e. population of cells at an early lineage stagethat is capable of expressing membrane-associated and/or secreted matrixmetalloproteinases (MMPs)

Not to be bound by theory, it is believed that that forms ofextracellular matrix found in amnions are able to keep the donor cellsimmature. Thus, amnions are contemplated both for use in the hydrogeland, optionally, as an alternative biocompatible, biodegradablematerial.

Notably materials known to cause maturation include certain componentsderived from mature extracellular matrix, such as but not limited totype I collagen. These materials should be excluded from all elements ofthe patch graft, including but not limited to the cells, the hydrogel,the medium, the backing, and/or any further components.

Thus, contemplated herein is a composition for use in the patch graftcomprising at least a population of cells and a source of MMPs (i.e. apopulation of cells at an early lineage stage that is capable ofexpressing membrane-associated and/or secreted matrix metalloproteinases(MMPs), supported in a suitable medium and comprised in a hydrogel.

As noted above, rigidity can drive the ability of cells todifferentiate. Further rigid hydrogels may have an effect on the abilityof cells to migrate. As the cells must migrate into the organ, thehydrogel in which the cells are comprised should have a viscoelasticitysufficient to allow for migration of said cells, optionally, within oraway from the hydrogel and/or the patch graft. Non-limiting examples ofsuch viscoelasticity include by are not limited a viscoelasticityranging from about 50 to about 100 Pa or about 250 Pa, for example atleast about 50 Pa, at least about 100 Pa, at least about 150 Pa, atleast about 200 Pa, at most about 250 Pa, at most about 200 Pa, at mostabout 150 Pa, at most about 100 Pa, and/or any individual value inbetween such as but not limited to about 50 Pa, about 100 Pa, about 150Pa, about 200 Pa, and about 250 Pa.

Not to be bound by theory, it is believe that when the cells migratefrom the patch graft into the target organ or tissue, they migrate withsome of the hydrogel associated with them or coating them. The hydrogelshields the cells from the signals in the tissue microenvironment whichwould influence the cells to differentiate or mature, and enables thecells to remain immature. This facilitates the cells migrating throughthe parenchymal tissue. As the hyaluronans in the hydrogel gradually getdegraded and removed, the cells begin to differentiate or mature andbegin adult cell functions.

Methods of Generating Organoids

Not to be bound by theory, it has been determined that early stagelineage cells may have a high rate of graft success when incorporatedinto an organoid or an aggregate. Such organize may optionally compriseearly lineage stages of both epithelial and of mesenchymal cells.

Thus, provided herein is a method of forming organoids, the methodcomprising, consisting of, or consisting essentially of culturing amixture of epithelial cells and mesenchymal cells in a containersuitable for tissue culture and in the presence of a culture medium,removing mature cells that attach to a surface of the container bypanning, and recovering self-assembled organoids from the suspension ofcells in the culture media. Also disclosed herein is a compositioncomprising an organoid generated as such.

In some embodiments, the procedure involves panning to eliminate maturecells by selective, rapid (15-30 minutes) attachment of them to regularculture dishes under serum-free conditions and at 37° C., since evenunder these conditions, the mature cells express various matrixcomponents that enable cell attachment. Multiple rounds (e.g. 4-5) ofsuch a panning process enriches the cell suspension for the earlierlineage stage cells. Then the cell suspension is transferred to lowattachment dishes and again in serum-free medium, one designed for theearly lineage stage cells, and left overnight in an incubator at 37° C.The conditions foster self-assembly of the lineage-stage-matchedepithelial and mesenchymal cells into organoids. Organoids can beobtained from mixing of early stages of epithelia (ES cells, iPS cells,determined stem cells, transit amplifying cells, progenitors) with earlystages of mesenchymal cells (angioblasts, precursors to endothelia,precursors to stellate cells).

Mixtures of adult epithelial cells with mature mesenchymal cells andchimeric mixtures of mature epithelial cells with early lineage stagemesenchymal cells (ELSMCs) do not usually generate organoids but can beused as mixtures of the cells in suspension in the graft biomaterials.If mature epithelia (e.g. hepatocytes, cholangiocytes, islets, acinarcells, enterocytes, etc.) are partnered with mature mesenchymal cells(e.g., endothelia, stellate cells, stromal cells, myofibroblasts), themixtures will not result in successful integration of the grafts intothe target site or organ but rather in ones that persist at the surfaceof the organs or tissues. If chimeric mixtures are used comprising adultand stem/progenitors (e.g. mature hepatocytes with angioblasts), thenengraftment does occur, since there is a source of MMPs that enableengraftment and migration of the cells.

In another aspect, the isolated cells of the internal organ may besolidified ex vivo within the biomaterials prior to introducing thecells into the hosts, or in the alternative, injected as a fluidsubstance and allowed to solidify into a graft in vivo. Preferably, thecells are introduced at or near the diseased or dysfunctional tissue,and may be introduced via injection or grafted onto/into the tissue, orusing an appropriate surgical method.

In another aspect, the biomaterials that can form hydrogels, or aparallel insoluble complex, can comprise hyaluronans, thiol-modifiedhyaluronans or other glycosaminoglycans (GAGs). A trigger forsolidification can be any factor eliciting cross-linking of the matrixcomponents or gelation of those components that can gel. Thecross-linker may comprise Poly(ethylene glycol) (PEG) or PEG-diacrylate(PEGDA) hydrogel or a disulfide-containing derivative thereof.

In another aspect, this disclosure provides a methods of formingorganoids by culturing a first type of cells (epithelia) with one ormore second type of cells (mesenchymal cells), wherein the second typeof cells is at a maturational stage to be an appropriate lineage partnerof the first type of cells. In some embodiments, this can be achieved byremoving mature cells that attach to culture dishes by panning;transferring the cells that did not attach to low attachment culturedishes and in an appropriate medium; and recovering organoids thatself-assemble under these conditions. The first type of cells may beepithelial stem cells, committed progenitors of epithelial cells, ormature cells (e.g. hepatocytes). The second type of cells may be stemcells of the mesenchymal lineages (e.g. angioblasts, mesenchymal stemcells), progenitors of those lineages (e.g. endothelial or stellate cellprogenitors), or a mixture of early lineage stage mesenchymal cells.Critically, such formation cannot occur under all conditions. Forexample, culturing in Matrigel does not generate suitable organoids forsuccessful patch grafting. Though Matrigel-prepared organoids mightengraft, the extent of engraftment will be muted relative to that withorganoids prepared in defined conditions. Moreover, Matrigel cannot be acomponent of conditions that are to be used for clinical products.

In another aspect, this disclosure provides a method for engraftingcells into an organ comprising contacting a patch graft comprisingmultiple layers including a biocompatible, biodegradable backing that isneutral in effects on the differentiation of the donor cells; a secondlayer comprising one or more biomaterials, such as hyaluronans, that canbe solidified such as into a hydrogel; a mixture of epithelial cells andsupportive mesenchymal cells that are incorporated into the solidifiedbiomaterial; and this Bandaid-like structure attached to a target siteby sutures or surgical glue. On the serosal surface of the backing isadded a layer of the solidified biomaterials prepared to achieve 400 Paor higher, a level at least twice that found in the soft biomaterialsinto which the donor cells are incorporated. The cells within the patchgraft are able to engraft and migrate into and throughout thetissue/organ and then to mature to relevant adult fates, dictated by themicroenvironment in which they become located. The higher Pascal levelsof the biomaterials embedded or comprised into a porous backing blocksthe migration of the cells in the wrong direction and that added to theserosal surface of the graft minimizes adhesions of cells from otherorgans and tissues.

Organoids

According to one embodiment disclosed herein, organoids, floatingaggregates of biliary tree stem cells (hereinafter “BTSCs”) and earlylineage stage mesenchymal cells (hereinafter “ELMCs”) proved the mostsuccessful method of incorporating cells in the grafts. It is disclosedherein that BTSCs and ELMCs can self-select into organoids by panning toeliminate the mature stellate/stromal cells, and this a proved moreefficient and effective in establishing lineage-stage appropriateepithelial-mesenchymal partners for the grafts. In another aspect, thisdisclosure provides a methods of forming organoids by culturing a firsttype of cells with a second type of cells, wherein the second type ofcells is a stage appropriate lineage partner of the first type of cells,removing mature cells that attach to the culture dish by panning, andrecovering the self-assembled organoids from the suspension of theculture. The first type of cells may be epithelial stem cells orcommitted epithelial cells. The second type of cells may be cells of themesenchymal lineage, mesenchymal stem cells, or early lineage stagemesenchymal cells. Further aspects relate to the self-assembled organoidand uses thereof.

In some embodiments, either the donor cells and/or the supportingmesenchymal cells express matrix metallo-proteinases (hereinafter MMPs).Without being limited by theory, it is believed that the MMPs allows formerger of donor and host cells, and the dissolution of Glisson's capsule(or the equivalent capsule around the tissue or organ). The disclosureherein provides that in some embodiments, the early stage stem cells orELMCs express high levels of MMPs, whereas the mature hepatocytesexpress low levels of MMPs. In some embodiments, partnering maturehepatocytes with mature sinusoidal endothelia (CD31+++, VEGF-receptor+,type IV collagen+ and negative for CD117) and those for adultcholangiocytes are associated with mature stellate and stromal cells(ICAM-1+, ASMA+, Vitamin A++, type I collagen+) results in cellaggregates that remain at the surface of the organ and cannot beeffectively engrafted. In some embodiments, engraftment of matureepithelial cells requires that they are partnered with immaturemesenchymal cells that produce the requisite MMPs for engraftment andmigration.

According to one embodiment disclosed herein, organoids, floatingaggregates of stem/progenitor cells, such as BTSCs and ELSMCs, provedthe most successful presentation of cells for success at patch grafting.It is disclosed herein that BTSCs and ELSMCs can self-select intoorganoids by elimination of the mature mesenchymal cells by standardpanning procedures for cells that attach to regular dishes underserum-free conditions, followed by culturing the remaining cells (thosethat did not attach) in low attachment dishes and in serum-free, definedmedium. Organoids self-assemble under these conditions.

In another aspect, this disclosure provides a method of formingorganoids by culturing a first type of cells, epithelia, with a secondtype of cells, mesenchymal cells, wherein the second type of cells is astage appropriate lineage partner of the first type of cells, removingmature cells that attach to the regular culture dishes by panningprocedures, and recovering the organoids that self-assemble from thesuspension of the culture on culture dishes that are low attachmentones. The first type of cells may be epithelial stem cells, transitamplifying cells committed epithelial progenitors. The second type ofcells may be stem cells of the mesenchymal cell lineages, transitamplifying cells or committed mesenchymal progenitors.

In some embodiments, either the donor cells and/or the supportingmesenchymal cells express matrix metallo-proteinases (hereinafter MMPs).Without being limited by theory, it is believed that the MMPs results indissolution of the capsules around tissues or organs and allows formerger of donor and host cells. The disclosure herein provides that insome embodiments, the early stage stem cells or ELMCs express highlevels of MMPs, whereas the mature hepatocytes express low levels ofMMPs. In some embodiments, partnering mature hepatocytes with maturesinusoidal endothelia (CD31+++, VEGF-receptor+, type IV collagen+ andnegative for CD117) and those for adult cholangiocytes are associatedwith mature stellate and stromal cells (ICAM-1+, ASMA+, Vitamin A++,type I collagen+) results in cell aggregates that remain at the surfaceof the organ and cannot be effectively engrafted. In some embodiments,engraftment of mature epithelial cells requires that they are partneredwith immature mesenchymal cells that produce the requisite MMPs forengraftment and migration.

According to one embodiment disclosed herein, organoids, floatingaggregates of stem/progenitor cells, such as BTSCs and ELSMCs, provedthe most successful presentation of cells for success at patch grafting.It is disclosed herein that BTSCs and ELSMCs can self-select intoorganoids by elimination of the mature mesenchymal cells by standardpanning procedures for cells that attach to regular dishes underserum-free conditions, followed by culturing the remaining cells (thosethat did not attach) in low attachment dishes and in serum-free, definedmedium. Organoids self-assemble under these conditions.

In another aspect, this disclosure provides a method of formingorganoids by culturing a first type of cells, epithelia, with a secondtype of cells, mesenchymal cells, wherein the second type of cells is astage appropriate lineage partner of the first type of cells, removingmature cells that attach to the regular culture dishes by panningprocedures, and recovering the organoids that self-assemble from thesuspension of the culture on culture dishes that are low attachmentones. The first type of cells may be epithelial stem cells, transitamplifying cells committed epithelial progenitors. The second type ofcells may be stem cells of the mesenchymal cell lineages, transitamplifying cells or committed mesenchymal progenitors.

In some embodiments, for success with patch grafting strategies, eitherthe donor cells and/or the supporting mesenchymal cells must expressmultiple matrix metallo-proteinases (hereinafter MMPs) and especiallysecreted forms of MMPs. Without being limited by theory, it is believedthat multiple isoforms of the MMPs allows for the dissolution of thecapsule around the organ or tissue followed by rapid migration of donorcells into the host tissue. The disclosure herein provides that theearly stage epithelial stem cells and/or ELSMCs express high levels ofmembrane-associated and/or secreted MMPs, whereas the mature cells (e.g.hepatocytes) express low levels of secreted MMPs even if they expressplasma membrane-associated MMPs. Engraftment of such adult cells (e.g.hepatocytes, cholangiocytes, islets, enterocytes, etc.) requires thatthe mesenchymal partner be a cellular source of MMPs, particularly thesecreted forms of MMPs if engraftment is to occur. An alternative is toprovide the relevant isoforms of MMPs, that is purified forms of them,in the biomaterials of the graft.

According to this disclosure, the numbers of cells that can be engraftedusing a patch graft are considerable (>10⁸) and dictated by thedimensions of the graft, the number and size of the organoids (or thenumber of cells—if not part of organoids), whether the donor cells arestem cells or mature cells, and the expression of secreted andmembrane-associated MMPs (whether from the epithelia and/or from themesenchymal cells). These findings are quite distinct from the limitednumbers of cells (e.g. 10⁵-10⁶) feasible with vascular delivery or byinjection grafting.

It is disclosed herein that the making the grafts comprises mixing ofcells with appropriate biomaterials that can become insoluble and keepcells localized to the target site. In another aspect, the isolatedcells of the internal organ may be solidified ex vivo within thebiomaterials prior to introducing the cells into the hosts, or in thealternative, injected as a fluid substance and allowed to solidify invivo. In another aspect, the biomaterials that can form hydrogels, or aparallel insoluble complex, can comprise hyaluronans or othernon-sulfated or minimally sulfated glycosaminoglycans, a thiol-modifiedsodium hyaluronate or plant derived material (e.g. alginates). A triggerfor solidification can be any factor eliciting cross-linking of thematrix components or gelation of those that can gel. The cross-linkermay comprise polyethylene glycol diacrylate or a disulfide-containingderivative thereof. Preferably, the insoluble complex of cells andbiomaterials possesses a viscoelasticity ranging from about 0.1 to 200Pa, preferably about 0.1 to about 1 Pa, about 1 to about 10 Pa, about10-100 Pa, or about 100 to about 200.

Preferably, the cells are introduced at or near the diseased ordysfunctional tissue, and may be introduced via injection or surgicaldelivery. Without being limited by theory, it is an hypothesis hereinthat more rigid HA hydrogels, (e.g. >500 Pa), triggers differentiationof the cells and reduces engraftment due, in part, to the reduction inexpression of MMPs with maturation and, in parallel reduction in abilityto migrate.

Backing

There are multiple options for the biocompatible, biodegradable backingwith neutrality to the maturational state of the donor cells. Theyinclude forms of Bombyx moth silk such as Seri® Surgical Silk Scaffoldsor Contour Seri-Silk (Sofregen, New York, N.Y.), other derivatives ofBombyx moth silk, amnion derivatives, omentum, placenta, and synthetictextiles or materials, such as forms of Polyglycolicacid-co-poly-L-lactic acid (PGA/PLLA). Critical to the effectiveness ofthe backing is that it has minimal effects on the differentiation of thedonor cells. Thus, many forms of backings used clinically are not usefulfor patch grafting, since they are comprised of components (e.g. formsof mature types of extracellular matrix) that induce differentiation ofthe donor cells.

The backing must have sufficient tensile strength to permit attachingthe graft to the target site by sutures or by surgical glue. It shouldbe comprised of a biocompatible, biodegradable material that is capableof degrading within a couple of months but with degradation productsthat do not alter the maturational state of the donor cells. Thus, theproducts should have minimal effects on the pH or on other facets of theenvironment. The backing must also be able to fit to the surface of thetarget site; so flexible backing will facilitate using the grafts onsites of significant curvature. Seri-Silk is a non-limiting example of asuitable material for the backing. An aminion derived alternative isalso contemplated as a suitable material for the backing, such as butnot limited to the aminion derived material produced by OsirisTherapeutics, Inc (Columbia, Md.).

Backing may be sourced from a porous scaffold, such as Seri-silk, or anon-porous membrane, such as amnion or placental membrane or omentum, orcan be a porous or non-porous synthetic textile, or a combinationthereof. If the backing is porous it should be infused/impregnated witha biomaterial to seal it and so inhibit migration of said population ofcells in the direction of the backing, i.e. away from the target site,or through the backing The critical features of the backing materialthat it is biocompatible, biodegradable, neutral as defined above, andhas sufficient tensile strength as described above. Further, thematerial may optionally be bioresorbable.

The backing may be further optimized depending on the use. For example,in some embodiments, a patch graft is useful for skin and underlyingdermal tissues if it comprises a backing designed to survive the dryingeffect of air.

Hydrogel matrices as disclosed herein above may also be useful in otherparts of the patch graft. For example, should the biocompatible,biodegradable backing be porous, a hydrogel may be used to inhibitmigration of said population of cells in the direction of the backing.Such a hydrogel would require a higher viscoelasticity compared to thehydrogel, e.g., between 1.5 and 15 fold greater, for example 2 foldgreater. Non-limiting examples of a suitable viscoelasticity include byare not limited a viscoelasticity properties ranging from about 250 toabout 600 Pa, for example at least about 250 Pa, at least about 300 Pa,at least about 350 Pa, at least about 400 Pa, at least about 450 Pa, atleast about 500 Pa, at least about 550 Pa, at most about 600 Pa, at mostabout 550 Pa, at most about 500 Pa, at most about 450 Pa, at most about400 Pa, at most about 350 Pa, at most about 200 Pa and/or any individualvalue in between such as but not limited to about 250 Pa, about 300 Pa,about 350 Pa, about 400 Pa, about 450 Pa, about 500 Pa, about 550 Pa,and about 600 Pa. Further non-limiting examples of suitableviscoelasticity include by are not limited a viscoelasticity rangingfrom about 600 to about 800 Pa, for example at least about 600 Pa, atleast about 650 Pa, at least about 700 Pa, at least about 750 Pa, atmost about 800 Pa, at most about 750 Pa, at most about 700 Pa, at mostabout 650 Pa, at most about 600 Pa, and/or any individual value inbetween such as but not limited to about 600 Pa, about 650 Pa, about 700Pa, about 750 Pa, and about 800 Pa. Still further non-limiting examplesinclude the range from about 250 Pa to about 800 Pa.

Further still, the hydrogels disclosed herein may be useful as a coatingto prevent adhesion on the serosal surface of the backing, which isopposite to the side of the backing adjacent to the cells. Such ahydrogel may should have a viscoelasticity between that suitable for thehydrogel in which the cells are comprised and that suitable to seal thebacking. Non-limiting examples of a suitable viscoelasticity include byare not limited a viscoelasticity ranging from about 250 to about 400 Paor about 500 Pa, for example at least about 250 Pa, at least about 300Pa, at least about 350 Pa, at least about 400 Pa, at least about 450 Pa,at most about 500 Pa, at most about 450 Pa, at most about 400 Pa, atmost about 350 Pa, at most about 200 Pa and/or any individual value inbetween such as but not limited to about 250 Pa, about 300 Pa, about 350Pa, about 400 Pa, about 450 Pa, and about 500 Pa.

Grafts, In General

In general, a patch graft may be designed using the aforementionedmethods and components for transplantation of donor (allogeneic orautologous) cells to a solid organ or tissue and with conditionssustaining and maintaining donor cells at an early maturational lineagestage. More particularly, a patch graft is contemplated, which usefulfor transplantation of donor cells (allogeneic or autologous) to a solidorgan or tissue, with conditions sustaining and maintaining some or allof the donor cells at an early maturational lineage stage. In someembodiments, the donor cells are a mixture of epithelial and mesenchymalcells. In some embodiments both donor cell populations arestem/progenitor cells. In some embodiments, the epithelial cells aremature cells (e.g. hepatocytes, islets, etc.) and the mesenchymal cellsare stem/progenitor cells. In some embodiments, the conditions of thegraft biomaterials, e.g. the medium and matrix components, enable boththe donor cell populations or at least the mesenchymal cell populationto remain as stem/progenitor cells. In some embodiments the mediumcomprises a basal medium and soluble signals. In further embodiments,this basal medium and soluble signals are supportive of maintenance ofstemness in both donor populations or at least in the mesenchymal cellpopulation. In some embodiments, the matrix, optionally comprisingextracellular matrix components, and its level of rigidity aresupportive of maintenance of stemness of both the donor populations orat least the mesenchymal cell population. In some embodiments, thematric comprises hyaluronans, optionally prepared as a soft hydrogelhaving a viscoelasticity of about 50 Pa to about 150 Pa. In someembodiments, the patch graft comprises a backing which has sufficientmechanical strength to enable the graft to be tethered to the targetsite and consists of a biocompatible, biodegradable material that doesnot significantly alter the maturational lineage stage of the donorcells. Optionally, without further modifications, the backing should beadequate on its own to protect the layer containing the donor cellswithout significantly affecting the donor cells' maturational lineagestage. In some embodiments, the backing is a mesh or scaffold and isfurther impregnated with a biomaterial such as hyaluronana with aviscoelasticity sufficiently high as to make any cells migrating into itmature enough to abrogate the migration of the donor cells in adirection other than towards the target site. In some embodiments, thisviscoelasticity is about 500 Pa or greater. In some embodiments, theserological surface of the graft is coated with a biomaterial tominimize adhesions from adjacent tissue or organs. In some embodiments,these biomaterials have a viscoelasticity of about 200 Pa to about 300Pa.

The proposed backing is contemplated to have sufficient resilience towithstand mechanical forces, is able to be tethered to the target organor tissue, and has sufficient flexibility to be tethered to locationswith curvature. Also any biomaterial (other than a hydrogel) can beutilized so long as the biomaterial is capable of sustaining andmaintaining the cell populations and has viscoelasticity propertiessufficient to allow for migration of the cell population within or awayfrom the patch graft.

In another embodiment, the patch graft is useful for sustaining andmaintaining a population of cells and comprises: (a) a population ofcells (optionally of a single type), supported in a medium in a hydrogelor other biomaterial having viscoelasticity sufficient to allow formigration of the cells within or away from the patch graft; and (b) abacking comprising a biocompatible, biodegradable material having aviscoelasticity sufficient to inhibit (or provide a barrier to)migration of the cell population in a direction of the backing,

It is important to note that MMPs can be membrane-associated and/orsecreted MMPs; they can be provided by MMP producing cells, derived fromsuch cells, or they can be added to the compositions of interest (e.g.,purified or produced recombinantly).

In another embodiment, a covering or coating for a patch graft or tissueis provided which comprises a hydrogel or other biomaterial withsufficient viscoelasticity and resilience to withstand mechanical forcesapplied against the covering or coating, including such forces beingapplied from or by other tissues and organs. By use of the covering orcoating, a method is provided for inhibiting or preventing a formationof adhesions (which may involve or result from mechanical forces orcontact from other organs and tissues), which method comprises coveringor coating a surface with a hydrogel or other comparable biomaterial.

In yet another embodiment, a method of engrafting cells into a targettissue is provided, which comprises contacting a target tissue with apatch graft, comprising: (a) a population of cells, including at leastone population having an early lineage stage, comprising a single typeor multiple types of cells supported in a medium in a hydrogel or otherbiomaterial having rheological properties (e.g., viscoelasticity)sufficient to allow for migration of cells of the population within oraway from the patch graft; and (b) a backing comprising a biocompatible,biodegradable material having rheological properties (e.g.,viscoelasticity) sufficient to inhibit (or provide a barrier to)migration of cells of the population in a direction of said backing, thepatch graft configured to sustain and maintain said population of cellswhile inhibiting said at least one population having an early lineagestage from differentiating or further maturing to a later lineage stage.In a further embodiment, a method is provided in which the onepopulation having an early lineage stage is capable of expressingmembrane-associated and/or secreted matrix metalloproteinases (MMPs). Inanother embodiment, the cells do not have this capability but MMPs arepresent or included from other sources (e.g. recombinant).

Grafts with a Cell Source of MMPs

Aspects of the disclosure relate to a patch graft for sustaining andmaintaining a mixed population of cells, comprising: (a) a mixedpopulation having two or more cell types, at least one of which is at anearly lineage stage that is capable of expressing secreted and/ormembrane-associated and/or secreted matrix metalloproteinases (MMPs),said mixed population supported in a medium present in a hydrogel matrixhaving a viscoelasticity sufficient to allow for migration of said mixedpopulation, optionally, within or away from said hydrogel and/or withinor away from the patch graft; (b) a backing comprising a biocompatible,biodegradable material having a viscoelasticity sufficient to inhibit amigration of said mixed population in a direction of said backing; and,optionally, ((c) a hydrogel overlaid on a serosal (i.e. outside) surfaceof said backing, which is opposite to that in contact with said mixedpopulation and, in embodiments where the patch graft is tethered to atarget site, is opposite the side in contact with the target site (e.g.organ or tissue). In some embodiments, this layer prevents or inhibitsadhesions by or from other tissues or organs. In some embodiments, thepatch graft is configured to sustain and maintain said mixed populationwhile inhibiting said at least one early lineage stage cell type fromdifferentiating or further maturing to a later lineage stage that is nolonger capable of expressing membrane-associated and/or secreted MMPs.

In some embodiments, the graft might contain only one cell type such asan embryonic stem (ES) cell or induced pluripotent stem (iPS) cells.This can be successful as long as this cells are a cellular source ofMMPs or, alternatively, other sources such as purified (e.g.recombinant) forms of MMPs are added to the graft.

In some embodiments, said backing is porous or non-porous. In someembodiments, the backing comprises a porous and/or non-porous mesh,scaffold, or membrane. In some embodiments, the backing comprises silk;a synthetic textile; or a natural material such as amnion, placenta, oromentum or derivatives thereof; or a combination thereof. In someembodiments, said backing comprises a porous mesh infused with ahydrogel or other biomaterial used to convert it into a barrier. Infurther embodiments, such an infusion prevents cell migration away fromthe target organ or tissue. In some embodiments, In some embodiments,said backing comprises a solid material.

In some embodiments, one or more of said hydrogels comprise hyaluronans.

In some embodiments, said medium comprises Kubota's Medium or anothermedium supportive of stem cells and able to maintain stemness.

In some embodiments, said mixed population comprises mesenchymal cellsand epithelial cells. In some embodiments, said epithelial cells may beectodermal, endodermal, or mesodermal. In some embodiments, saidmesenchymal cells comprise early lineage stage mesenchymal cells(ELSMCs). In some embodiments, said ELSMCs comprise one or more ofangioblasts, precursors to endothelia, precursors to stellate cells, andmesenchymal stem cells (MSCs). In some embodiments, said epithelialcells comprise epithelial stem cells. In some embodiments, saidepithelial cells comprise biliary tree stem cells (BTSCs). In someembodiments, said epithelial cells comprise committed and/or matureepithelial cells. In some embodiments, said committed and/or matureepithelial cells comprise mature parenchymal cells. In some embodiments,said mature parenchymal cells comprise one or more of hepatocytes,cholangiocytes, and islet cells. In some embodiments, said mesenchymalcells and epithelial cells both comprise stem cells.

In some embodiment said mixed population comprises autologous and/orallogeneic cells.

In some embodiments, one or more cell types are genetically modified.

“Layered” Grafts

In some embodiments, the patch graft is understood as a multi-layeredgraft. For example, provided herein are patch grafts comprising,consisting of, or consisting essentially of multiple layers including,at least: (a) a soft first layer of hydrogel comprising donor cells,optionally epithelial cells and/or mesenchymal cells; (b) a stiff secondlayer of hydrogel; and (c) a third layer comprising a biocompatible,biodegradable backing. In some embodiments, particular those where thethird layer is porous, the second layer is incorporated, impregnated,and/or infused into the third layer. In some embodiments, the patchgrafts further comprise a fourth layer of hydrogel. In some embodimentsof the patch graft, the fourth layer is coated or painted onto a serosalsurface of the graft. In some embodiments of the patch graft, the firstlayer is adapted to directly contact a target tissue or organ.

As used herein, “soft” refers to a hydrogel layer that exhibits a lowlevel of internal pressure as determined quantitatively by Pascal (Pa)assays. A Pascal is defined as one newton per square meter. In someembodiments, a soft layer has a viscosity of about 10 Pa to about 300Pa, about 50 Pa to about 250 Pa, about 100 Pa to about 250 Pa, about 50Pa to about 200 Pa, about 150 Pa to about 200 Pa, or about 100 Pa toabout 200 Pa. In a particular embodiment, a soft hydrogel layer has aviscosity that is less than or about 200 Pa.

As used herein, “stiff” refers to a hydrogel layer that exhibits a highlevel of internal pressure as determined quantitatively by Pascal (Pa)assays. In some embodiments, a stiff layer has a viscosity of about 300Pa to about 3000 Pa, about 300 Pa to about 1000 Pa, about 400 Pa toabout 750 Pa, about 400 Pa to about 550 Pa, about 450 Pa to about 600Pa, or about 500 Pa to about 600 Pa. In a particular embodiment, a stiffhydrogel layer has a viscosity that is greater than or about 500 Pa.

Preferably, for the first layer of the layered graft, the insolublecomplex of cells and biomaterials possesses a viscosity orviscoelasticity ranging from about 0.1 to 200 Pa, preferably about 0.1to about 1 Pa, about 1 to about 10 Pa, about 10 to 100 Pa, or about 100to about 200, or about 50 to about 250 Pa, or about 200 Pa. Preferably,for the first layer of the layered graft, the insoluble complex of cellsand biomaterials possesses a viscoelasticity ranging from about 0.1 to200 Pa, preferably about 0.1 to about 1 Pa, about 1 to about 10 Pa,about 10-100 Pa, or about 100 to about 200.

In some embodiments, one or more of the cells in the mixture is a sourceof secreted and/or membrane-associated MMPs. In some embodiments, suchas but not limited to those involving stem/progenitor cell populationsthat naturally secrete MMPs, variables that mute MMPexpression—optionally secreted MMP expression—are controlled in thepatch graft. Non-limiting examples of such variables include variablesthat result in maturation of stem/progenitor cells, such as but notlimited to serum supplementation to media or to the graft biomaterials,hormones or other soluble signals that influence differentiation of theepithelial and/or mesenchymal cells, oxygen levels (as anaerobicconditions keep the cells immature, whereas higher oxygen levels promotedifferentiation), and the rigidity of graft materials (as mechanicalforces such as shear force and compression may drive differentiation).

In some embodiments of the patch graft, the viscosity of the first layeris about 50 to about 250 Pa. In some embodiments of the patch graft, theviscosity of the first layer is about 200 Pa. In some embodiments of thepatch graft, the viscosity of the second layer is about 250 Pa to about600 Pa. In some embodiments of the patch graft, the viscosity of thesecond layer is about 500 Pa. In some embodiments of the patch graft,the viscosity of the fourth layer is about 250 to about 500 Pa. In someembodiments of the patch graft, the viscosity of the fourth layer isabout 400 Pa. In some embodiments of the patch graft, the viscosity ofthe second layer is greater than the viscosity of layer 1. In someembodiments of the patch graft, the viscosity of the second layer isabout 1.5 to about 15 fold greater than the viscosity of the firstlayer. In some embodiments of the patch graft, the second layer is about2 fold greater than the viscosity of the first layer.

In one embodiment, a patch graft comprises, consists of, or consistsessentially of layers starting with that in contact with the target siteand consisting of donor cells embedded into a soft (<200 Pa) hydrogelprepared in a serum-free, defined medium (these cells are to engraft andmigrate into the tissue); a second layer of the hydrogel prepared in thesame medium and triggered to have a higher rigidity (e.g. ˜500 Pa orhigher) providing a barrier for the donor cells to migrate in anydirection other than towards the target tissue; a third layer, abiocompatible, biodegradable, bioresorable backing that is neutral ineffects on the maturational state of the donor cells and can be usedsurgically or through other means to tether the graft to the targetsite; and a final layer of the hydrogel that is intermediate in rigiditybetween the soft hydrogel and the very rigid one and sufficiently fluidto be painted or coated onto the surface to minimize adhesions by nearbytissues.

In some embodiments of the patch graft, the first and second layers eachcomprise one or more hyaluronans. In some embodiments of the patchgraft, the fourth layer comprises one or more hyaluronans.

In some embodiments of the patch graft, the epithelial cells and themesenchymal cells form one or more aggregates. In some embodiments ofthe patch graft, the one or more aggregates is an organoid. In someembodiments of the patch graft, the epithelial cells comprise epithelialstem cells. In some embodiments of the patch graft, the epithelial cellscomprise biliary epithelial cells. In some embodiments of the patchgraft, the epithelial cells comprise committed and/or mature epithelialcells. In some embodiments of the patch graft, the committed and/ormature epithelial cells comprise mature parenchymal cells. In someembodiments of the patch graft, the mature parenchymal cells compriseone or more of hepatocytes, cholangiocytes, and islet cells.

In some embodiments of the patch graft, the mesenchymal cells aresupportive mesenchymal cells. In some embodiments of the patch graft,the mesenchymal cells comprise early lineage stage mesenchymal cells(ELSMCs). In some embodiments of the patch graft, the ELSMCs compriseone or more of the group consisting of angioblast, precursor toendothelia, precursor to stellate cells, and mesenchymal stem cell(MSC).

In some embodiments of the patch graft, the epithelial cells and themesenchymal cells are not lineage stage partners of one another. In someembodiments of the patch graft, the epithelial cells are mature cells.In some embodiments of the patch graft, the mesenchymal cells areELSMCs.

In some embodiments of the patch graft, at least one of the epithelialcells and the mesenchymal cells are derived from a donor. In someembodiments, the donor is a subject in need of a tissue transplant. Insome embodiments, the donor is the source of healthy cells for a tissuetransplant. In some embodiments of the patch graft, the at least one ofthe epithelial cells and the mesenchymal cells are autologous to anintended recipient of the patch graft. In some embodiments, all of thecells (i.e. epithelial and mesenchymal) are autologous to the intendedrecipient of the graft. In some embodiments, the donor of cells may beone other than the recipient (allograft) or may also be the subject(autologous) having the internal organ in a diseased or dysfunctionalcondition, optionally, wherein are obtained from a portion of theinternal organ that is not diseased or dysfunctional and/or that thecells have been genetically modified to restore function. Forestablishing a model system to study a disease, the donor cells can beones that have the disease and that are transplanted onto/into normaltissue in an experimental host.

In some embodiments of the patch graft, at least one of the epithelialcells or the mesenchymal cells are modified. In some embodiments, all ofthe cells are modified. In some embodiments, the modification is geneticmodification. In some embodiments, the one or more cells is modified toexpress a therapeutic nucleic acid or polypeptide. In some embodiments,the one or more cells is modified to express a wild-type allele of anucleic acid or polypeptide.

In some embodiments of the patch graft, the biocompatible, biodegradablebacking is bioresorbable. In some embodiments of the patch graft, thebiocompatible, biodegradable backing comprises a porous material. Insome embodiments of the patch graft, the biocompatible, biodegradablebacking comprises a scaffold or membrane. In some embodiments of thepatch graft, the scaffold or membrane comprises silk, amnion, asynthetic textile, or a combination thereof. In some embodiments, thebiocompatible, biodegradable backing does not comprise any factor thatinduces or prevents differentiation in cells. In some embodiments of thepatch graft, the biocompatible, biodegradable backing does not includeone or more components derived from mature extracellular matrix. In someembodiments of the patch graft, the component derived from matureextracellular matrix is type I collagen.

In some embodiments of the patch graft, the patch graft furthercomprises one or more matrix metallo-proteinases (MMPs). In someembodiments of the patch graft, the MMP is a membrane-associated MMP. Insome embodiments of the patch graft, the membrane-associated MMP isprovided by one or more of the epithelial cells or the mesenchymalcells. In some embodiments of the patch graft, the MMP is a secretedMMP. The secreted MMPs may optionally be produced naturally by the oneor more of the epithelial or mesenchymal cells or optionally be produceddue to transformation of the one or more of the epithelial ormesenchymal cells with a recombinant expression vector for MMPproduction.

In some aspects, provided herein is a patch graft comprising, consistingof, or consisting essentially of multiple layers including, at least: asoft first layer of hydrogel comprising biliary tree stem cells; a stiffsecond layer of hydrogel; and a third layer comprising a biocompatible,biodegradable backing.

In one embodiment, a patch graft consists of layers of materials andcells that collectively form a “bandaid-like graft” that can be tetheredsurgically or otherwise to a target site. The first layer, that againstthe target site, comprises a soft hydrogel (under 200 Pa) into which areseeded a mixture of epithelial cells and supportive mesenchymal cellssuspended in a defined, serum-free, nutrient-rich medium designed forexpansion and/or survival of the cells; a second layer containing ahydrogel prepared in the same medium but gelled to a more rigid level(i.e. higher Pascal levels) and forming a barrier blocking cells frommigrating in a direction other than to the target sites; a third levelcomprising a biocompatible, biodegradable backing that does not affector minimally affects the differentiation level of the donor cells butacting as a mechanical support structure for the patch; a fourth layercomprised of paintable hydrogel (again such as hyaluronans) that is at arigidity level intermediate between that of the soft versus rigidhydrogel and serving to minimize adhesions to the graft from cells fromneighboring tissues. The hydrogels must consist of a material that isbiocompatible, biodegradable and “tunable”, meaning regulatable withrespect to rigidity. One successful material for the hydrogels isthiol-modified hyaluronan that can be triggered to form hydrogels whenexposed to oxygen and/or to poly (ethylene glycol) diacrylate (PEGDA)and readily “tunable” by the precise ratios of hyaluronan and PEGDAconcentrations (and/or oxygen levels).

In another embodiment, a patch graft comprises multiple layers. Thefirst layer, that against the target site, is of a soft hydrogel that isminimally sulfated or non-sulfated GAG or other non-sulfated or neutralbiomaterial that can be gelled or solidified and into which is placeddonor cells. A second layer of a hydrogel or biomaterial that is morerigid and incorporated into/onto or within a backing, a biocompatible,biodegradable, bioresorbable backing that allows the patch to be handledfor surgical or other purposes and that serves as a barrier forcingcells to migrate towards the target tissue. The serosal side of thebacking is coated at the time of surgery with biomaterials such ashyaluronans (or other minimally or non-sulfated GAGs or other materialsthat can be gelled or solidified) and in which the Pascal levels are atleast twice that of the Pascal levels found in the layer of softbiomaterials; this serves the purpose of minimizing adhesions fromneighboring tissues. The patch graft is tethered to the target organ ortissue, and the cells are able to migrate into the tissue or organ andbecome fully incorporated.

In a particular embodiment, a patch graft comprises a first layer of asoft biomaterial (<200 Pa), such as a soft hyaluronan hydrogel, and intowhich are placed the donor cells to be transplanted in a serum-free,defined medium tailored to the lineage stage of the cells. This layer isplaced atop a more rigid layer (e.g. a more rigid hydrogel) that servesas a barrier forcing the donor cells to be directed in their migrationto the target tissue. The more rigid layer is prepared ahead of time ona backing, a biocompatible, biodegradable backing that enables handlingthe patch for surgical or other procedures so as to affix the patch tothe target site. The final layer is a biomaterial that is intermediatein rigidity from that for the donor cells on the target tissue side andthat for the barrier. This layer is added on the serosal side of thegraft and at the time of surgery and serves to minimize adhesions fromneighboring tissues. The biocompatible, biodegradable backing may beSeri-silk or a derivative thereof.

Methods of Use and Delivery for Patch Grafts

Aspects of the disclosure relate to compositions and methods forengrafting cells into an organ. Efforts to transplant cells from solidorgans into internal organs typically made use either of directinjection or delivery of cells via a vascular route. Lanzoni, G. et al.Stem Cells 31, 2047-2060 (2013). These methods of transplantation resultin small numbers of cells being transplanted to the target site, and inrisks of emboli that can be life threatening. Transplantation isimproved if the cells are delivered by “injection grafting” in which thecells are suspended in or coated with hyaluronans and then co-injectedwith a trigger (PEGDA) that causes the hyaluronan to gel in situ asdescribed in Turner R. et al. Hepatology 57, 775-784 (2013). Injectiongrafting methodologies provide a strategy for localizing cells to aspecific site, albeit in small numbers, typically 10⁵-10⁷, 10⁶-10⁷, or10⁵-10⁶ cells per injection site. This strategy eliminates or minimizesectopic cellular distribution and optimizes the integration of the cellsin the site. However, if mature functional cells are used, they may behighly immunogenic, necessitating long-term immunosuppression. Also, thequantity of cells that are able to be injected may be insufficient toachieve the requisite clinical results.

These hurdles and concerns are overcome by “patch grafting” strategiesdescribed herein. In some embodiments, “bandaid-like” grafts aretethered surgically or otherwise to the surface of an organ or tissue;the conditions of the graft are such that the cells engraft fully intothe site, migrate throughout the organ/tissue, and then mature intorelevant adult cell types. The potential for transplantation of largenumbers of cells (>10⁸ cells) is shaped or determined by the size of thepatch, the number or mixture of cells within the graft, and the sourceof multiple forms of MMPs, ideally cellular sources of the MMPs.Moreover, in some embodiments the use of organoids facilitates theability to stockpile donor cells given the ease by which the organoidscan be cryopreserved under defined, serum-free conditions.

The patch graft composition provided herein is directed to directgrafting of cells onto the tissue or solid organ. The method is safe,avoids emboli and ectopic cell distribution, and optimizes cell numberengraftment and distribution into and throughout the tissue.

Accordingly, provided herein are methods of engrafting cells into atarget tissue comprising, consisting of, or consisting essentially ofcontacting the target tissue with a patch graft disclosed herein above.

In some embodiments of the methods, the target tissue is selected fromthe group consisting of liver, pancreas, biliary tree, thyroid, thymus,gastrointestine, lung, prostate, breast, brain, bladder, spinal cord,skin and underlying dermal tissues, uterus, kidney, muscle, bloodvessel, heart, cartilage, tendons, and bone tissue. In some embodimentsof the methods, the target tissue is liver tissue. In some embodimentsof the methods, the target tissue is pancreatic tissue. In someembodiments of the methods, the target tissue is biliary tree tissue. Insome embodiments of the methods, the target tissue is gastrointestinaltissue. In some embodiments, the tissue is diseased, damaged, or has adisorder. In some embodiments of the methods, the target tissue iskidney tissue.

In some embodiments of the methods, the target tissue is an organ. Insome embodiments of the methods, the organ is an organ of themusculoskeletal system, the digestive system, the respiratory system,the urinary system, the female reproductive system, the malereproductive system, the endocrine system, the circulatory system, thelymphatic system, the nervous system, or the integumentary system. Insome embodiments of the methods, the organ is selected from the groupconsisting of liver, pancreas, biliary tree, thyroid, thymus,gastrointestines, lung, prostate, breast, brain, bladder, spinal cord,skin and underlying dermal tissues, uterus, kidney, muscle, bloodvessel, heart, cartilage, tendon, and bone. In some embodiments, theorgan is diseased, damaged, or has a disorder.

Also provided herein are methods of treating a subject with a liverdisease or disorder, the methods comprising, consisting of, orconsisting essentially contacting the subject's liver a patch graftdisclosed herein above. In some embodiments of the methods, the liverdisease or disorder is liver fibrosis, liver cirrhosis, hemochromatosis,liver cancer, biliary atresia, nonalcoholic fatty liver disease,hepatitis, viral hepatitis, autoimmune hepatitis, fascioliasis,alcoholic liver disease, alpha 1-antitrypsin deficiency, glycogenstorage disease type II, transthyretin-related hereditary amyloidoisis,Gilbert's syndrome, primary biliary cirrhosis, primary sclerosingcholangitis, Budd-Chiari syndrome, liver trauma, or Wilson disease.

In other aspects, provided herein are methods of treating a subject witha disease or disorder of the pancreas, the methods comprising,consisting of, or consisting essentially of contacting the subject'spancreas with a patch graft disclosed herein above. In some embodimentsof the methods, the disease or disorder of the pancreas is diabetesmellitus, exocrine pancreatic insufficiency, pancreatitis, pancreaticcancer, sphincter of Oddi dysfunction, cystic fibrosis, pancreasdivisum, annular pancreas, pancreatic trauma, or hemosuccuspancreaticus.

In other aspects, provided herein are methods of treating a subject witha gastrointestinal disease or disorder, the method comprising,consisting of, or consisting essentially of contacting one or more ofthe subject's intestines with a patch graft disclosed herein above. Insome embodiments, the gastrointestinal disease or disorder isgastroenteritis, gastrointestinal cancer, ileitis, inflammatory boweldisease, Crohn's disease, ulcerative colitis, irritable bowel syndrome,peptic ulcer disease, celiac disease, fibrosis, angiodysplasia,Hirschsprung's disease, pseudomembranous colitis, or gastrointestinaltrauma.

In some aspects, provided herein are methods of treating a subject witha kidney disease or disorder, the methods comprising, consisting of, orconsisting essentially of contacting one or more of the subject'skidneys with a patch graft disclosed herein above. In some embodimentsof the methods, the kidney disease or disorder is nephritis, nephrosis,nephritic syndrome, nephrotic syndrome, chronic kidney disease, acutekidney injury, kidney trauma, cystic kidney disease, polycystic kidneydisease, glomerulonephritis, IgA nephropathy, lupus nephritis, kidneycancer, Alport syndrome, amyloidosis, Goodpasture syndrome, or Wegener'sgranulomatosis.

In some embodiments of the therapeutic methods, at least one of theepithelial cells and the mesenchymal cells are derived from a donor. Insome embodiments, the donor is a subject in need of a tissue transplant.In some embodiments, the donor is the source of healthy cells for atissue transplant. In some embodiments, at least one of the epithelialcells and the mesenchymal cells are autologous to an intended recipientof the patch graft. In some embodiments, all of the cells (i.e.epithelial and mesenchymal) are autologous to the intended recipient ofthe graft. In some embodiments, the donor of cells may be one other thanthe recipient (allograft) or may also be the subject (autologous) havingthe internal organ in a diseased or dysfunctional condition, optionally,wherein are obtained from a portion of the internal organ that is notdiseased or dysfunctional and/or that the cells have been geneticallymodified to restore function.

In some embodiments, the patch graft used in the methods disclosedherein above is a patch graft comprising multiple layers including, atleast: a first layer of hydrogel comprising epithelial cells andmesenchymal cells; a second layer of hydrogel; a third layer comprisinga biocompatible, biodegradable backing; and optionally a fourth layer ofhydrogel. In some embodiments, the methods further comprise allowing thecells contained in the patch graft to become incorporated into thetissue. In some embodiments of the methods, the first layer of hydrogelis soft. In some embodiments of the methods, the second layer ofhydrogel is stiff. In some embodiments of the methods, the mesenchymalcells are supportive mesenchymal cells.

In another aspect, this disclosure provides a method for engraftingcells into an organ comprising use of a patch graft, a bandaid-likecomposite with multiple layers of materials and cells that collectivelycan be tethered surgically or otherwise to a target site. The firstlayer, that against the target site, comprises a soft hydrogel (under200 Pa) into which are seeded a mixture of epithelial cells andsupportive mesenchymal cells suspended in a defined, serum-free,nutrient-rich medium designed for expansion and/or survival of thecells; a second layer containing a hydrogel prepared in the same mediumbut gelled to a more rigid level (i.e. higher Pascal levels) and forminga barrier blocking cells from migrating in a direction other than to thetarget sites; a third level comprising a biocompatible, biodegradablebacking that does not affect or minimally affects the differentiationlevel of the donor cells and hence is “neutral;” a fourth layercomprised of paintable hydrogel (again such as hyaluronans) that is at arigidity level intermediate between that of the soft versus rigidhydrogel and serving to minimize adhesions to the graft from cells fromneighboring tissues. The hydrogels must consist of a material that isbiocompatible, biodegradable and “tunable”, meaning regulatable withrespect to rigidity. One successful material for the hydrogels isthiol-modified hyaluronan that can be triggered to form hydrogels whenexposed to oxygen and/or to poly (ethylene glycol) diacrylate (PEGDA)and readily “tunable” by the precise ratios of hyaluronan and PEGDAconcentrations (and/or oxygen levels). The cells under the conditions ofthe biomaterials of the graft produce multiplematrix-metallo-proteinases (MMPs) that facilitate engraftment,migration, and integration of the donor cells into the tissue of therecipient. The microenvironment of the recipient tissue dictates theadult fate of the transplanted cells.

In another aspect, this disclosure provides a method for engraftingcells into an organ comprising contacting a patch graft comprisingmultiple layers including, at least, a first layer comprising abiocompatible, biodegradable backing, a second layer comprising one ormore hyaluronans including a mixture of epithelial cells and supportivemesenchymal cells and a third layer comprising one or more hyaluronans,in which the layer in which the cells are embedded is very soft (under200 Pa); a layer associated with the backing is more rigid (˜500 Pa ormore); and a third layer is intermediate in the level of Pascals andhelps to minimize adhesions from nearby tissues or organs. In yetanother aspect, the cells may be engrafted into an organ selected fromthe group consisting of liver, pancreas, biliary tree, thyroid, thymusintestines, lung, prostate, breast, brain, spinal cord, neural ganglia,skin and underlying dermal tissues, uterus, bone, thymus, intestines,uterus, bone, kidney, muscle, blood vessels, or heart.

In yet another aspect, the cells may be engrafted into an organ selectedfrom the group consisting of liver, pancreas, biliary tree, thyroid,thymus thymus, intestines, lung, prostate, breast, brain, spinal cord,neural ganglia, skin and underlying dermal tissues, uterus, bone,tendon, cartilage, kidney, muscle, blood vessels, or heart.

A non-limiting example of a patch graft suitable for the methodsdisclosed herein is a patch graft comprising: (a) a mixed populationhaving two or more cell types, at least one of which is at an earlylineage stage that is capable of expressing secreted and/ormembrane-associated and/or secreted matrix metalloproteinases (MMPs),said mixed population supported in a medium present in a hydrogel matrixhaving a viscoelasticity sufficient to allow for migration of said mixedpopulation, optionally, within or away from said hydrogel and/or withinor away from the patch graft; (b) a backing comprising a biocompatible,biodegradable material having a viscoelasticity sufficient to inhibit orprovide a barrier to migration of said mixed population in a directionof said backing; and, optionally, ((c) a hydrogel overlaid on a serosal(i.e. outside) surface of said backing, which is opposite to that incontact with said mixed population and, in embodiments where the patchgraft is tethered to a target site, is opposite the side in contact withthe target site (e.g. organ or tissue). In some embodiments, this layerprevents or inhibits adhesions by or from other tissues or organs. Insome embodiments, the patch graft is configured to sustain and maintainsaid mixed population while inhibiting said at least one early lineagestage cell type from differentiating or further maturing to a laterlineage stage that is no longer capable of expressingmembrane-associated and/or secreted MMPs.

In some embodiments, said backing is porous or non-porous. In someembodiments, the backing comprises a porous mesh, scaffold, or membrane.In some embodiments, the backing comprises silk; a synthetic textile; ora natural material such as aminion, placenta, or omentum; or acombination thereof. In some embodiments, said backing comprises aporous mesh infused with a hydrogel. In further embodiments, such aninfusion prevents cell migration away from the target organ or tissue.In some embodiments, In some embodiments, said backing comprises a solidmaterial.

In some embodiments, the patch graft further comprises a hydrogeloverlaid on a serosal surface of said backing, which is opposite to thatin contact with said single cell or mixed cell population.

In some embodiments, one or more of said hydrogels comprise hyaluronans.

In some embodiments, said medium comprises Kubota's medium or anothermedium supportive of stem cells and able to maintain stemness.

In some embodiments, said mixed population comprises mesenchymal cellsand epithelial cells.

In some embodiments, said epithelial cells may be ectodermal,endodermal, or mesodermal. In some embodiments, said mesenchymal cellscomprise early lineage stage mesenchymal cells (ELSMCs). In someembodiments, said ELSMCs comprise one or more of angioblasts, precursorsto endothelia, precursors to stellate cells, and mesenchymal stem cells(MSCs). In some embodiments, said epithelial cells comprise epithelialstem/progenitor cells. In some embodiments, said epithelial cellscomprise biliary tree stem cells (BTSCs). In some embodiments, saidepithelial cells comprise committed and/or mature epithelial cells. Insome embodiments, said committed and/or mature epithelial cells comprisemature parenchymal cells. In some embodiments, said mature parenchymalcells comprise one or more of hepatocytes, cholangiocytes, and isletcells. In some embodiments, said mesenchymal cells and epithelial cellsboth comprise stem cells.

In some embodiment said mixed population comprises autologous and/orallogeneic cells.

In some embodiments, one or more cell types are genetically modified.

EXAMPLES

The following examples are non-limiting and illustrative of procedureswhich can be used in various instances in carrying the disclosure intoeffect. Additionally, all reference disclosed herein below areincorporated by reference in their entirety.

Example 1: Porcine Model for Patch Graft Validation

Animals

Animals used as hosts or as donors for cells were maintained infacilities at the College of Veterinary Medicine at NCSU (Raleigh,N.C.). Surgeries, necropsies, and the collection of all biologicalfluids and tissues were performed at these facilities. All procedureswere approved by the IACUC committee at NCSU. The pigs being used asrecipients were a mixture of six different breeds: a six-way crossconsisting of Yorkshires, Large Whites, Landraces (from the sows),Durocs, Spots, and Pietrans (from the boars). This highly heterogeneousgenetic background is desirable in that it parallels the heterogeneousgenetic constitutions of human populations. The host animals were allfemales, approximately six weeks of age and ˜15 kg.

There were two categories. a) male pigs, approximately six weeks of ageand ˜15 kg, were used as donors for cell transplantation into females;b) transgenic donor animals carrying a GFP transgene. The GFP+ donoranimals were obtained by breeding a transgenic H2B-GFP boar with a wildtype gilt by standard artificial insemination. The model was developedvia CRISPR-Cas9 mediated homology-directed repair (HDR) ofIRES-pH2B-eGFP into the endogenous β-actin (ACTB) locus. The transgenicanimals show ubiquitous expression of pH2B-eGFP in all tissues. Fusionof the GFP to H2B results in localization of the GFP marker to thenucleosome and allows clear nuclear visualization as well as the studyof chromosome dynamics. The founder line has been analyzed extensivelyand ubiquitous and nuclear localized expression has been confirmed. Inaddition, breeding has demonstrated transmission of the H2B-GFP to thenext generation. All animals were healthy, and multiple pregnancies havebeen established with progeny showing the expected Mendelian ratio forthe transmission of the pH2B-eGFP. The male offspring were genotyped atbirth, and those that were positive for the transgene were humanelyeuthanized for tissue collection, and isolation of donor cells.

For each donor and recipient animal, the swine leucocyte antigen class I(SLA-I) and class II (SLA-II) loci have been PCR amplified using primersdesigned to amplify known alleles in these regions based on thePCR-sequence-specific-primer strategy. The system consists of 47discriminatory SLA-I primer sets amplifying the SLA-1, SLA-2, and SLA-3loci⁵³, and 47 discriminatory SLA-II primer sets amplifying the DRB1,DQB1, and DQA loci. These primer sets have been developed todifferentiate alleles by groups that share similar sequence motifs, andhave been shown easily and unambiguously to detect known SLA-I andSLA-II alleles. When used together, these primer sets effectivelyprovided a haplotype for each animal that was tested, thus providing anassay to confirm easily a matched or mismatched haplotype in donor andrecipient animals.

Media and Solutions

All media were sterile-filtered (0.22 μm filter) and kept in the dark at4° C. before use. Basal medium and fetal bovine serum (FBS) werepurchased from GIBCO/Invitrogen. All growth factors were purchased fromR&D Systems. All other reagents, except those noted, were obtained fromSigma.

A cell wash was formulated with 599 mls of basal medium (e.g. RPMI 1640;Gibco #11875-093) supplemented with 0.5 grams of serum albumin (Sigma, #A8896-5G, fatty-acid-free), 10-9 M selenium, and 5 mls of antibiotics(Gibco #35240-062, AAS). It was used for washing tissues and cellsduring processing.

Collagenase buffer was made and consists of 100 mls of cell washsupplemented with collagenase (Sigma # C5138) with a final concentrationof 600 U/ml (R1451 25 mg) for biliary tree (ducts) tissue and 300 U/ml(12.5 mg) for organ-parenchymal tissue (liver, pancreas).

Kubota's medium, a defined, serum-free medium designed for endodermalstem/progenitors was used to prepare cell suspensions, organoids and HAhydrogels. This medium consists of any basal medium (here being RPMI1640) with no copper, low calcium (0.3 mM), 1 nM selenium, 0.1% bovineserum albumin (purified, fatty-acid-free; fraction V), 4.5 mMnicotinamide, 0.1 nM zinc sulfate heptahydrate, 5 μg/ml transferrin/Fe,5 μg/ml insulin, 10 μg/ml high density lipoprotein, and a mixture ofpurified free fatty acids that are presented complexed with fatty acidfree, highly purified albumin. Its preparation is given in detail in amethods review⁵⁷. Also, it is available commercially from PhoenixSongsBiologicals (Branford, Conn.).

Soluble, long chain forms of HA (Sigma Catalog #52747) were used instabilization of organoid cultures and in cryopreservation Those used tomake the hydrogels, thiol-modified HAs, were obtained from GlycosanBiosciences, a subsidiary of Biotime. The components for thesethiol-modified HAs were made by a proprietary bacterial-fermentationprocess using bacillus subtilis as the host in an ISO 9001:2000 process(www.biopolymer.novozymes.com/). The components were produced byNovozymes under the trade name HyaCare® and are 100% free ofanimal-derived raw materials and organic solvent remnants. Noanimal-derived ingredients are used in the production, and there arevery low protein levels and no endotoxins. The production follows thestandards set by the European Pharmacopoeia) The HA hydrogels wereprepared using Glycosil (HyStem® HAs, ESI BIO-CG313), the thiol-modifiedHAs, that can be trigged to form disulfide bridges using polyethyleneglycol diacrylate (PEGDA). Glycosil® is reconstituted as a 1% solutionof thiolated HA in 1% phosphate buffered saline (PBS) using degassedwater, or, in our case, in Kubota's Medium. Upon reconstitution, itremains liquid for several hours but can undergo some gelation ifexposed to oxygen. More precise gelation occurs with no temperature orpH changes if Glycosil is treated with a cross-linker such as PEGDAcausing gelation to occur within a couple of minutes.

The level of cross-linking dictates the level of rigidity, and can beprecisely defined by the ratio of the thiol-modified HAs to PEGDA. Inprior studies, stem cell populations were tested in HA hydrogels ofvarying level of rigidity and were found to remain as stem cells, bothantigenically and functionally (e.g. with respect to ability tomigrate), only if the level of rigidity was less than 200 Pa²³. We madeuse of this finding to design the grafts with a very soft layer and withmore rigid layers of hyaluronan hydrogels on the serosal side to form abarrier to migration in directions other than the target tissue as wellas to minimize adhesions from cells from nearby tissues. The 3 versionsof the hydrogels with distinct levels of rigidity are characterized inFIG. 2, characterizations that included direct measurements of therheological properties. The most rigid barrier, that of the 10×HAhydrogel (rigidity=760 Pa), was prepared on the backing ahead of timeand could be cryopreserved if desired. At the time of the surgery, thedonor cells were prepared in the soft, 1×HA hydrogel (rigidity=60 Pa);placed onto the more rigid 10× hydrogel (already on the backing); andthe patch tethered to the target site. After tethering, the serosal sideof the graft was coated or painted with the 2×HA hydrogel (rigidity=106Pa) using a NORM-JECT 4010.200V0 Plastic Syringe with a BD Micro-Fine™IV permanently attached needle.

Macro-scale rheological properties of hydrogels were determined using astress-controlled cone-and-plate rheometer (TA Instruments, AR-G2, 40 mmcone diameter, 1° angle). Gels actively polymerized on the rheometerwhile oscillating at 1 rad/s frequency and 0.6 Pa stress amplitude withthe modulus monitored continuously to query for sufficient completion ofthe cross-linking reaction. Once equilibrated, the hydrogels weresubjected to an oscillatory frequency sweep (stress amplitude: 0.6 Pa,frequency range: 0.01-100 Hz). The viscoelasticity (rheological)properties of the 3 versions of hyaluronan hydrogels that were used aresummarized in FIG. 2.

The most commonly used donor cells were derived from transgenic H2B-GFPpigs as described above. They offer a significant advantage for celltransplantation studies in that all cells are tagged with GFP. The useof fluorescent proteins as molecular tags enabled the donor cells to betracked in their migration and engraftment after transplantation. Thisfusion protein is targeted to the nucleosomes resulting in anuclear/chromatin GFP signal. In the described grafts, the stem cellsexpress GFP entirely in the nucleus, but those lineage restricting toadult cell types can have it in the cytoplasm or nucleus. Note that thelevel of cytoplasmic GFP is especially high in the first week and isreduced with time. This is because the engraftment/invasion/integrationprocess results in effects on the cells that can cause the H2B-linkedGFP to be found cytoplasmically. This does not mean that the cells aredying but rather that they are responding to the high levels of MMPs andassociated signaling that are part of the remodeling zones. Indeed, theGFP+ cells detected are clearly viable and proliferate, all expressingvarious adult functions (e.g. albumin, HNF4a, AFP, insulin, glucagon, oramylase).

As described in more detail in the characterizations of the grafts,autofluorescence both of the backing (spring green color) and also oflipofuscins (dark forest green color) in mature hepatocytes presented achallenge given the overlap in wavelengths with those of GFP. Therefore,Applicants shifted the GFP+ signal to a pink or rose color using anantibody to GFP and secondarily to an antibody with a red fluoroprobe.This resulted in the stem cells being recognized as small cells withpink nuclei (merger of the nuclear blue DAPI staining with theantibody-tagged-rose colored GFP+ label). Any donor cells that maturedinto hepatocytes were recognized as having a lavender color from themerger of the green autofluorescence (lipofuscins), the blue (DAPI), andthe rose-color (GFP) (FIG. 4).

Porcine extrahepatic biliary tree tissue (gall bladder, common duct,hepatic ducts) were obtained from transgenic pigs. Tissues were poundedwith a sterilized, stainless steel mallet to eliminate the parenchymalcells, carefully keeping the linkage of the intra-hepatic andextrahepatic bile ducts. The biliary tree was then washed with the “cellwash” buffer comprised of a sterile, serum-free basal mediumsupplemented with antibiotics, 0.1% serum albumin, and 1 nM selenium(10⁻⁹M). It was then mechanically dissociated with crossed scalpels, andthe aggregates enzymatically dispersed into a cell suspension inRPMI-1640 supplemented with 0.1% bovine serum albumin (BSA), 1 nMselenium, 300 U/ml type IV collagenase, 0.3 mg/ml deoxyribonuclease(DNAse) and antibiotics. Digestion was done at 32° C. with frequentagitation for 30-60 minutes. Most tissues required two rounds ofdigestions followed by centrifugation at 1100 rpm at 4° C. Cell pelletswere combined and re-suspended in cell wash. The cell suspension wascentrifuged at 30 G for 5 minutes at 4° C. to remove red blood cells.The cell pellets were again re-suspended in cell wash and filteredthrough a 40 μm nylon cell strainer (Becton Dickenson Falcon #352340)and with fresh cell wash. The cell numbers were determined and viabilitywas assessed using Trypan Blue. Cell viability above 90-95% wasroutinely observed.

In prior studies, Applicants have defined the antigenic profile ofpopulations of mesenchymal cells that provide critical paracrine signalsneeded for hepatic and biliary tree stem cells versus others requiredfor mature parenchymal cells. The mesenchymal cells that partner withBTSCs are subpopulations devoid of MEW antigens, with low side scatter,and identifiable as angioblasts (CD117+, CD133+, VEGF-receptor+, andnegative for CD31), precursors to endothelia (CD133+, VEGF-receptor+,and CD31+), and precursors to stellate cells (CD146+, ICAM1+, VCAM+,alpha-smooth muscle actin (ASMA)+, and negative for vitamin A). These 3subpopulations are referred to collectively as early lineage stagemesenchymal cells (ELSMCs). By contrast, adult hepatocytes areassociated with mature sinusoidal endothelia (CD31+++, type IVcollagen+, VEGF-receptor+, and negative for CD117) and those for adultcholangiocytes that are associated with mature stellate and stromalcells (ICAM-1+, ASMA+, Vitamin A++, type I collagen+).

The cell suspensions were added to Multiwell Flat Bottom Cell CulturePlates (Corning #353043) in serum-free Kubota's Medium and incubated for˜an hour at 37° C. to facilitate attachment of mature mesenchymal cells.Mature mesenchymal cells attached to the dishes within 10-15 minuteseven though the medium was serum-free. The cells remaining in suspensionwere transferred to another dish and again incubated for up to an hour.Repeats of this resulted in depletion of a significant fraction of themature mesenchymal cells. After depletion of mature mesenchymal cells,the remaining floating cells were seeded at ˜2×10⁵ cells per wells inserum-free Kubota's Medium in Corning's ultralow attachment dishes(Corning #3471) and were incubated overnight at 37° C. in a CO2incubator. Organoids comprised of the biliary tree stem cells (BTSCs)and of ELMSCs formed overnight (FIG. 1). These organoid culturessurvived for weeks in Kubota's Medium, especially if the medium wassupplemented (0.1%) with soluble forms of HAs (Sigma); they could alsobe cryopreserved as described below. From each gram of neonatal pigbiliary tree tissue, we obtained ˜1.5×10⁷ cells. We used ˜3-6×10⁵ cellsper well of a 6-well, ultra-low attachment plate and incubated in theserum-free Kubota's Medium. The cells produced, on average, 6000 to20,000 small organoids (˜50-100 cells/organoid/well). For the grafts, weused at least 100,000 organoids (>10⁷ cells). Depending on the size ofthe backing, Applicants were able to increase the number of organoids inthe grafts up to 10⁸ organoids (i.e. ˜10⁹ cells) or more embedded in ˜1ml of the soft hyaluronan hydrogel on a 3 cm×4.5 cm backing.

Isolated stem cell organoids were cryopreserved in CS10, an isotoniccryopreservation buffer containing antifreeze factors, dextran and DMSO(Bioliife, Seattle, Wash.;https://www.stemcell.com/products/cryostor-cs10.html). The viability ofthe cells was improved further with supplementation with 0.1% HAs (Sigma#52747). Cryopreservation was done using CryoMed™ Controlled-RateFreezers. The viability on thawing was greater than 90%, and cells afterthawing were able to attach, to expand ex vivo and in vivo and to giverise to the expected mature cells in vitro and in vivo.

Isolating the cells and assembling the grafts are characterized in aschematic in FIG. 1 and with the details summarized in FIG. 2. Thegrafts were formed by using a backing (TABLE 1) onto which were placedthe stem cell organoids embedded in the soft hyaluronan hydrogels. Thesewere readily prepared ahead of time and maintained in a culture dish inan incubator overnight. The grafts proved stable at the target site forthe duration of the experiments. Cryopreservation of the organoids wasachieved readily, but that of the organoids when within the softhydrogel was not. This meant that embedding the organoids in the softhydrogel had to be done just prior to surgery.

Surgeries

Anesthesia was induced by administering a combination ofketamine/xylazine (2-3 mg/kg weight each) injected IV or 20 mg/kgketamine plus 2 gm/kg xylazine IM, and was maintained by isoflurane inoxygen administered via a closed-circuit gas anesthetic unit.

The animals were positioned in dorsal recumbency, and the ventralabdomen was clipped from xyphoid to pubis. The skin was asepticallyprepared with alternating iodinated scrub and alcohol solutions. Afterentry into the surgery suite, preparation of the skin was repeated usingsterile technique, and the area was covered with a topical iodinesolution before application of sterile surgical drapes. The surgeonsused appropriate aseptic technique. A mid-ventral incision was madethrough the skin, through subcutaneous tissues and linea alba, startingat the xiphoid process and extending caudally 8-12 cm. The left hepaticdivision was exposed and a 3×4.5 cm patch graft was applied to theventral surface of the liver and containing 1×HA (˜60 Pa) with embeddedorganoids placed onto the backing containing 10×HA (˜760 Pa), and thepatch was placed in direct contact onto the surface of the livercapsule. The patch graft was sutured to the liver using 4-6 simple,interrupted sutures of 4-0 polypropylene. The exposed surface of thegraft was then treated with 2 mls of 2×HA hydrogel (˜106 Pa), a level ofrigidity that was fluid enough to permit it to be painted or coated ontothe serosal side of the graft; it served to further minimize adhesionsfrom neighboring tissues. Following placement of the surgical graft, thelinea alba was closed with a simple continuous suture using 0-PDS. Thelinea was blocked with 2 mg/kg 0.5% bupivacaine, IM. The subcutaneoustissues and skin were closed with continuous 2-0 PDS and 3-0 Monocrylsutures, respectively. Tissue adhesive was placed on the skin surface.

The graft transplants from the transgenic pigs to the recipients wereallogeneic and so required immunosuppression. The immune-suppressionprotocols used were ones established by others. All pigs received oraldosages of the immunosuppressive drugs Tacrolimus (0.5 mg/kg) andMycophenolate (500 mg) twice daily, beginning 24 hours prior to surgery.The drugs were given continuously for the entire experimental period.These could be given to the animals easily if mixed with their favoritefoods.

All animals were humanely euthanized at the designated time point bysedation with Ketamine/Xylazine, and isofluorane anesthesia, followed byan intravenous injection of a lethal dose of sodium pentobarbital. Uponconfirmation of death, the carcass was carefully dissected, and thetarget organs were removed, and placed in chilled Kubota's Medium fortransportation to the lab. In addition to the liver, the lungs, heart,kidney, and spleen were collected and fixed in 10% neutral formalin.

Characterization of the Grafts

After 48+ hours of fixation, tissues samples were placed in labeledcassettes in 70% ethanol and were processed on a long cycle at 60degrees in a Leica ASP300S Tissue Processor for approximately 10 hours.After completion of the overnight processing, samples were embeddedusing the Leica EG1160 Embedding Station. A mold was filled with wax andthe sample was placed in the correct orientation so that desiredsections could be collected. The cassette was chilled until the blockand tissue sample could be removed as one unit from the mold. The blockwas sectioned at 5 microns using a Leica RM2235 Microtome; the sectionswere floated in the water bath and placed onto slides. The slides wereallowed to air dry overnight before staining. Sections were stained forHaematoxylin and Eosin (H&E; Reagents #7211 and #7111) or Masson'sTrichrome (Masson's Trichrome Stain: Blue Collagen Kit#87019) usingRichard Allan Scientific Histology Products and following themanufacturer's recommended protocol; the protocol is programmed into aLeica Autostainer XL.

Tissue was embedded and frozen in OCT and flash frozen at −20° C. forfrozen sectioning. Frozen sections were stained for IHC followed theprotocol described above. For immunofluorescence, frozen sections werethawed for 1 hour at room temperature and then fixed in 10% bufferedformaldehyde, acetone or methanol according to the antibodyspecifications. After fixation, sections were washed 3 times in 1%phosphate buffered saline (PBS), followed by blocking with 2.5% horseserum in PBS for 1 hour at room temperature. Primary antibodies dilutedin 10% goat serum in PBS were added and incubated overnight at 4° C. Thenext morning, sections were rinsed 3 times with PBS and incubated withsecondary antibodies diluted in 2.5% horse serum in PBS for 2 hours atroom temperature. Images were taken using a Zeiss CLSM 710 SpectralConfocal Laser Scanning microscope (Carl Zeiss Microscopy). Antibodiesare listed in TABLE 3.

For the images in FIG. 5, sections (3 μm) were stained withhematoxylin-eosin and Sirius red, according to standard protocols. Forimmunohistochemistry, endogenous peroxidase activity was blocked by a 30min incubation in methanolic hydrogen peroxide (2.5%). Antigens wereretrieved, as indicated by the vendor, by applying Proteinase K (code53020, Dako, Glostrup, Denmark) for 10 min at room temperature. Sectionswere then incubated overnight at 4° C. with primary antibodies(pan-Cytokeratin, Dako, code: Z0622, dilution: 1:100; Sox9, Millipore,code: AB5535, dilution: 1:200). Samples were rinsed twice with PBS for 5min, incubated for 20 min at room temperature with secondarybiotinylated antibody (LSAB+ System-HRP, code K0690; Dako, Glostrup,Denmark) and then with Streptavidin-HRP (LSAB+ System-HRP, code K0690,Dako, Glostrup, Denmark). Diaminobenzidine (Dako, Glostrup, Denmark) wasused as substrate, and sections were counterstained with hematoxylin(PMID: 29248458). For immunofluorescence, non-specific protein bindingwas blocked by 5% normal goat serum. Specimens were incubated overnightat 4° C. with primary antibodies (chicken anti-GFP, Abcam, code:ab13970, dilution=1:200; rabbit anti-HNF4α, Abcam, code: 92378,dilution: 1:50, rabbit anti-albumin, ab2406, dilution=1:500). Specimenswere washed and incubated for 1 h with labeled isotype-specificsecondary antibodies (anti-chicken AlexaFluor-546, anti-mouseAlexafluor-488, anti-rabbit Alexafluor-488, Invitrogen, LifeTechnologies Ltd, Paisley, UK) and counterstained with4,6-diamidino-2-phenylindole (DAPI) for visualization of cell nuclei(PMID: 26610370). For all immunoreactions, negative controls (theprimary antibody was replaced with pre-immune serum) were also included.Sections were examined in a coded fashion by Leica Microsystems DM 4500B Light and Fluorescence Microscopy (Leica Microsystems, Weltzlar,Germany), equipped with a Jenoptik Prog Res C10 Plus Videocam (Jena,Germany). Immunofluorescence stains were also analyzed by ConfocalMicroscopy (Leica TCS-SP2). Slides were further processed with an ImageAnalysis System (IAS—Delta Sistemi, Roma—Italy) and were independentlyevaluated by two researchers in a blind fashion. Immunofluorescencestains were scanned by a digital scanner (Aperio Scanscope FL System,Aperio Technologies, Inc, Oxford, UK) and processed by ImageScope.

Frozen sections were problematic given the high autofluorescence inhepatocytes (lipofuscin) and the fluorescence of the Seri-Silk backing.Applicants had greater success by preparing paraffin sections andstaining for the GFP using a rabbit polyclonal antibody to GFP (NovusBiologicals, NE600-308); the rabbit anti-GFP antibody was used incombination with a secondary antibody of donkey anti-rabbit IgG H&L(Alexa Fluor 568; ab175470, Invitrogen), while Donkey anti-Goat IgGAlexa Fluor 488 antibody was used to exclude non-specific staining ofhepatic autofluorescence. Autofluorescence was reduced by quenching withthe use of dyes and that included Trypan Blue. The Trypan Blue was usedon tissues/cells at 0.4% in PBS. This reduces the backgroundsignificantly.

Total RNA was extracted from the organoids or grafts using Trizol(Invitrogen). First-strand cDNA synthesized using the Primescript 1ststrand cDNA synthesis kit (Takara) was used as a template for PCRamplification. Quantitative analyses of mRNA levels were performed usingFaststart Universal Probe Master (Roche Diagnostics) with ABI PRISM7900HT Sequence Detection System (Applied Biosystems). Primers weredesigned with the Universal Probe Library Assay Design Center (RocheApplied Science). Primer sequences are listed in TABLE 4. The primerswere annealed at 50° C. for 2 min and 95° C. for 10 min, followed by 40cycles of 95° C. (15 s) and 60° C. (1 min). Expression ofglyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used generally as acontrol and a standard.

RNA was purified from cells using the Qiagen RNeasy Kit RNA integrity(RIN) analysis was performed using an Agilent 2000 Bioanalyzer. The cDNAlibraries were generated using the Illumina TruSeq Stranded mRNApreparation kit and sequenced on the Illumina HiSeq 2500 platform. Twosamples were sequenced per lane, occupying a total of 8 lanes for all ofthe samples (one flow cell). Quality control analysis was completedusing FastQ. Mapping of sequence reads to the human genome (hg19) wasperformed with MapSplice2 using default parameters. Transcriptquantification was carried out by RSEM analysis, and DESeq was used tonormalize gene expression and identify differentially expressed genes.MapSplice2 was also used to detect candidate fusion transcripts. Fusioncalls were based on the depth and complexity of reads spanning candidatefusion junctions. Gene expression profiles were compared using Pearson'scorrelation analysis and hierarchical clustering was performed in R.Hierarchical clustering was performed following Variance StabilizingTransformation provided in the DESeq package. Pathway enrichmentanalysis was performed with the Ingenuity Pathway Analysis (IPA)software. Differential gene expression analysis was conducted only ongenes with a minimum average normalized count >50 in at least onecategory.

Statistically significant differences between samples were calculated byusing Student's 2-tailed t test and results are presented as themean±standard deviation (SD). P values of less than 0.05 were consideredstatistically significant.

Results

In prior studies on injection grafting, it was found that engraftmentrequired co-transplantation of epithelial cells with theirlineage-stage-appropriate mesenchymal cell partners. For hepatic andbiliary tree stem cells, these mesenchymal cells are comprised ofangioblasts (CD117+, CD133+, VEGFr+, CD31-negative) and their immediatedescendants, precursors to endothelia (CD133+, VEGFr+, CD31+, VanWillebrand Factor+) and precursors to stellate cells (CD146+, ICAM-1+,alpha-smooth muscle actin+(ASMA), vitamin A-negative). Applicants referto these collectively as early lineage stage mesenchymal cells (ELSMCs.Applicants also had partial success also with isolated porcinemesenchymal stem cells (MSCs) prepared by the methods of others andisolating cells from neonatal pig livers.

In prior studies, Applicants achieved isolating matching epithelial andmesenchymal cell stages by using multiparametric flow cytometry todetermine the ratios of the lineage stage partners of epithelial andmesenchymal cells in cell suspensions and then used those ratios withingrafts using immuno-selected cells. In these studies, Applicants foundit more efficient to deplete cell suspensions of mature mesenchymalcells by repeated panning procedures followed by culturing remainingcell suspensions on low attachment dishes and in serum-free Kubota'sMedium for 6-8 hours. Organoids self-assembled with each aggregatecontaining approximately 50-100 cells. Marker analyses indicatedpartnering of BTSCs with ELSMCs (FIG. 1). As summarized in the schematicin FIG. 1A, they were used immediately or were cryopreserved underdefined conditions determined previously and thawed as needed forgrafts. Organoids of BTSCs/ELSMCs were characterized usingimmunofluorescence (IF), qRT-PCR and RNA-seq and shown to expressclassic traits of BTSCs (FIG. 1) and of ELSMCs (data not shown). BTSCsin the organoids expressed no mature hepatic or pancreatic genes but lowlevels of pluripotency genes (e.g. OCT4, SOX2) and endodermal stem cellgenes (e.g. EpCAM, SOX 9, SOX17, PDX1, LGR5, CXCR4, MAFA, NGN3 and NIS).Representative qRT-PCR assays confirmed the findings from IF and fromIHC on cells prior to transplantation (FIG. 1D). IHC assays indicatedthat more primitive cells (e.g. ones expressing pluripotency genes) weredistributed to the interiors of the organoids and later maturationallineage stages at the perimeters (e.g. cells expressing EpCAM oralbumin) (FIG. 1C).

Results from patch grafts were compared with those from injection graftswith methods established previously and comprised of injection of cellsand with localization to the site by triggering hyaluronans withpolyethylene glycol diacrylate (PEGDA) to gel within minutes. Injectiongrafts into the porcine liver parenchyma resulted in essentially 100%engraftment but with minimal (if any) migration and with integrationinto the host tissue occurring slowly over weeks (data not shown). Thefindings were similar to those observed previously with injection graftsof hepatic stem cells¹⁷. Injection grafts into the mesentery adjacent tohepatic ducts/portal vein branches immediately caudal to the liver lobeswere feasible with large ducts but caused smaller ones to occlude fromthe swelling effects of HA hydrogels and resulting in cholestasis (FIG.13). Success with patch grafting led us to abandon further efforts withinjection grafting strategies.

The composition of the grafts for stem cells involved use of conditionswith 3 distinct layers of hyaluronans (HA) hydrogels with preciseconcentrations of HA to PEGDA to achieve a level of rigidity assessed byrheological assays (FIG. 2C). Donor cells were embedded into a soft HAlayer (˜100 Pa) and placed against the liver/pancreas surface; the softhydrogels maintained stemness traits²³ that in these studies provedessential for engraftment. This layer was placed on top of a rigid (10×;˜700 Pa) HA layer prepared ahead of time on the backing and serving as abarrier to migration. The patch was attached to the target site withsutures or surgical glue. A 2×HA hydrogel, soft enough (rigidity=˜200Pa), to permit painting or coating the serosal surface of the graft atthe time of the surgery and serving to further minimize adhesions fromnearby tissues.

Patch grafts were placed onto the liver surface, i.e. superficial to theGlisson capsule or pancreatic capsule, and attached by sutures or bysurgical glue at the corners (FIG. 2F). The stiffness of Seri-Silkresulted in grafts being placed at sites with minimal curvature and awayfrom sites with significant mechanical forces (e.g. near the diaphragm).In the grafts onto pancreas, the graft was wedged between the duodenumand the pancreas.

The only variant of patch grafting attempted and then abandoned wasafter sharp surgical removal of the capsule. Hemorrhaging was excessiveobviating future use in hosts with altered hemostasis associated withhepatic failure or even in normal hosts given the adverse influences ofserum on donor cells. Without such efforts to alter the organ capsules,patch grafts proved facile for surgical procedures.

A number of backings were tried with a focus on ones used clinically inabdominal surgeries (TABLE 1 and TABLE 2). All but Seri-Silk causedproblems that resulted in their elimination for further consideration.The problems included fragility (e.g. Seprafilm, Retroglyde); inductionof necrosis or fibrosis and significant levels of adhesions (e.g.Surgisis, Vetrix); and severe adhesion formations with a filamentoussponge version of Seri-Silk or any of the backings supplemented withcarboxymethylcellulose (“belly jelly”) to the abdomen. Of the onestested, SERI Surgical Silk²⁴⁻²⁶ (Allergan, Inc. Irvine, Calif.) providedthe best combination of mechanical support and minimal adhesions, aneffect further enhanced by application of 2×HA to the serosal surface ofSeriSilk after attachment to the target site. The product is a purifiedfibroin of Bombyx moth silk and was developed by David Kaplan (Tuft'sUniversity, Boston, Mass.). Applicants found it to be stiff, a propertyfound useful for surgical manipulations and placement on flat/rigidorgans like the liver. The stiffness made it difficult to apply to siteswith significant curvature or need for flexibility. Still, its stiffnessproved neutral with respect to maturational effects on the donor cells,a finding that made this backing acceptable for patch grafting. Ingrafts at 3 weeks, Seri-Silk was enveloped by bands of collagen,suggesting a mild foreign body reaction. Assessment of other candidatebackings, such as synthetic textiles, is ongoing.

Evidence for remodeling at week one after surgery was validated withTrichrome staining (FIGS. 3, 7) or Safranin O, having dyes that staincollagens and other extracellular matrix components. The images of thegraft (FIG. 3A-B) that are stained with Trichrome are compared with onesof the same site and stained with hematoxylin/eosin (FIG. 3 C-D).Reconstitution of the Glisson capsule and of the lobules occurred by 3weeks in parallel with HAs being resorbed. The bands comprising the areaof remodeling were surprisingly large (FIGS. 3-5, 7).

Donor cells deriving from transgenic GFP+ pigs were identified readilyby GFP expression through IHC assays. In pancreas, the donor cells wereidentified by the green fluorescence. However, in liver, theautofluorescence of the lipofuscins in hepatocytes peaks at a wavelengthoverlapping with that for GFP. Therefore, we identified donor cells inlivers with an antibody to GFP (Rabbit anti-GFP antibody; Novus,NB600-308) and coupled to a secondary antibody with a red fluoroprobe(Donkey anti-rabbit 555, Invitrogen) causing donor cells to have pinknuclei (the red fluoroprobe plus the blue DAPI). Host cells wererecognized given their blue nuclei (DAPI stain) but without GFPexpression (FIG. 4).

The liver lobules of mature hepatocytes were forest-green from theautofluorescence (lipofuscins) (FIG. 4B). Donor GFP+ cells that hadmatured to aggregates of hepatocytes were a lavender color and with pinknuclei (FIG. 4C) due to the merger of the red fluoroprobe from GFP, theblue from DAPI, and the autofluorescent dark green from lipofuscins.Hepatocytes, whether host or donor derived, were clustered around byhost mesenchymal cells (endothelia, stellate cells) with brightyellow/green autoflorescence due, we assume, to vitamin A in the maturestellate cells (FIG. 4C); the IHC data for the endothelia and stellatecells are not shown.

Within a week, patch grafts of BTSCs/ELSMCs organoids resulted inremodeling of the organ capsule and adjacent lobules followed by amerger of host and donor cells (FIGS. 3-5, 7). Finger-like extensions ofdonor cells extended into the hepatic lobules of the host tissue; inparallel, host cells extended into HAs of the grafts (FIG. 4). In thecase of the pancreas, the graft was wedged between the pancreas and theduodenum, and by one week post surgery, engraftment of donor cellsoccurred both into the pancreas and into the Brunner's glands of thesubmucosa of the duodenum (FIG. 6). Integration of the cells withinlarge regions of the liver (or the pancreas) was completed by 2 weeks bywhich time the layers of HAs had been mostly resorbed; donor cells hadlineage restricted into adult hepatic parenchymal fates, bothcholangiocytic and hepatocytic (FIG. 5) or into pancreatic fates (FIG.6).

By 3 weeks, the HA layers were resorbed entirely, leaving only thebacking. This correlated with reappearance of the organ capsule and ofthe histological structure of the tissue near to the capsules (FIGS. 3,5, 6) or of the pancreatic capsule and of the pancreatic histologicalstructures (FIG. 6). In pancreas, mature cells were identified byfunctional markers that included insulin for islet cells (beta cells)and amylase for acinar cells.

Engraftment efficiency for both the liver and for the pancreas was closeto 100% by a week, since all donor cells identified were found to beviable and within the liver or pancreas; not in the remnants of thegrafts above the organ capsules; and with negligible or no evidence ofectopic cell distribution in other organs (e.g. lung).

The speed of migration of donor cells in the BTSC/ELSMC grafts throughthe liver and through the pancreas proved remarkable resulting in donorcells in most regions of the organ (liver or pancreas) by the end of aweek and with uniformly dispersed cells throughout the tissue(liver/pancreas) by 2-3 weeks (FIGS. 3-6).

Correlated with the dissolution and remodeling of the Glisson capsule(or pancreatic capsule) and neighboring liver lobules (or pancreatictissue) and correlating with significant engraftment was elevatedexpression of multiple MMPs, enzymes known to dissolve extracellularmatrix components and to be associated with cell migration. In FIG. 7are summarized data from RNA-seq studies and IHC assays on MMPsexpressed by stem/progenitors versus adult cells. BTSCs expressed highlevels of multiple MMPs, comprised of both secreted forms (e.g. MMP2,MMP7) as well as membrane-associated forms (e.g. MMP14 and MMP15). TheELSMCs, precursors of endothelia and of stellate cells, also contributedto multiple MMPs.

The findings from RNA-seq data were confirmed by IHC assays for theproteins encoded by MMP genes (FIG. 7). IHC assays confirmed thepresence of the secreted forms of MMPs (e.g. MMP1, MMP2, MMP7, MMP9)especially in the regions of remodeling. Protein expression of MMP1 wasfound in BTSCs/ELSMCs organoids and also in remodeling regions ofgrafts; however, existing data banks of RNA-seq findings do not includeMMP1 because of a lack of an annotated species of porcine MMP1 to beused for the analyses. Therefore, recognition of its expression is basedon IHC assays.

Variables causing differentiation of donor cells resulted in a muting ofexpression of MMPs, especially the secreted forms, and, in parallel, aloss in potential for engraftment and migration (data not shown). Thesefactors included serum, various soluble regulatory signals (growthfactors, cytokines, hormones) known to influence differentiation of thedonor cells, extracellular matrix components whether in the hydrogels orin the backings (especially type I collagen-containing backings), andthe stiffness of the HA hydrogels (i.e. the Pa levels). Ifdifferentiation of the ELSMCs progressed preferentially to stroma, thegrafts became fibrotic; if to endothelia, the grafts retained viablecells and tissue but remained superficial to the organ capsule (data notshown).

Organoids of BTSCs/ELSMCs proved the most successful arrangement for thecells for grafting. In the past, we had co-transplantedepithelial-mesenchymal partners by immuno-selecting them from cellsuspensions by flow cytometry using their distinctive surface antigens,and then mixing them according to the ratios found in cells suspensionsfrom freshly isolated tissues¹⁷. Here we found that letting themself-select into organoids, after removal by panning of maturemesenchymal cells, proved more efficient and effective in establishinglineage-stage appropriate epithelial-mesenchymal partners with relevantparacrine signaling for the grafts and yielding organoids under defined(serum-free) conditions, that made them easily and safely cryopreserved.

The primary design of the grafts consisted of mixing of cells withappropriate biomaterials that can become insoluble and keep cellslocalized to the target site. For grafts, the ideal biomaterials provedto be non-sulfated or minimally sulfated glycosaminoglycans (GAGs), suchas hyaluronans (HAs), found in all stem cell niches, with receptors toHAs being classic stem cell traits. Maintenance of cells asstem/progenitors optimized expression of secreted andmembrane-associated MMPs effective for engraftment.

Evidence of engraftment processes was particularly dramatic withinregions of remodeling that occurred at the interface of the graft andthe host tissue. To validate the findings of remodeling, Trichromestaining and Safranin O were used having dyes that stain extracellularmatrix components and analyzed in parallel with adjacent sectionsstained with hematoxylin/eosin (FIG. 3, 7). It confirmed remodeling ofthe organ capsule and of adjacent tissue within a week after surgery. By3 weeks post-surgery, these assays demonstrated reconstitution of theorgan capsules and of the normal tissue histology following clearance ofHAs. The remodeling zone was surprisingly large (FIG. 3, 7), especiallyat one week after surgery and was shown to involve multiple forms ofMMPs (FIG. 7)

Although there are many sources and types of HAs, among the most usefulare thiol-modified ones established by Glenn Prestwich (University ofUtah, Salt Lake City, Utah) and that can be triggered with PEGDA to forma hydrogel with precise biochemical and mechanical properties. Theseproperties of HAs confer perfect elasticity, allow access into the graftof all soluble signals in blood, lymph or interstitial fluid, andminimize the maturation of donor cells until engraftment and migrationhave occurred. The ability to vary the rheological factors with simplechanges in HA and PEGDA concentrations provided additional advantages inguiding the direction of migration of the cells and in minimizingadhesions. Soft HA hydrogels, ones mimicking properties in stem cellniches, were permissive for expression of the stem/progenitorcell-associated repertoire of MMPs. Thus, the mechanical properties ofHAs, studied for years in the functions of skeletal tissues, areimportant also in managing grafting strategies²³.

Patch grafts containing stem/progenitors resulted in a strikingphenomena of grafts “melting” into tissues within a few days, followedby a merger of donor and host cells, and a distribution of cellsthroughout most regions of the organ by one to two weeks. Thereafter,maturation of donor cells and restoration of the organ capsules occurredin parallel with the tissue clearance of HAs.

The engraftment and integration process correlated with expression ofmultiple MMPs, a family of calcium-dependent, zinc-containingendopeptidases that degrade extracellular matrix components. UsingRNA-seq studies, we found a pattern of stem/progenitor-associated MMPs,comprised of high levels of secreted forms (e.g. MMP2, MMP7) as well asmembrane-associated forms (e.g. MMP14, MMP15). IHC assays indicated thatprotein levels of secreted MMPs (e.g. MMP1, MMP2, MMP7) were foundrichly expressed in areas of remodeling (FIG. 7). Conditions (solublegrowth factors, cytokines, serum, matrix components, mechanical forces)that caused donor cells to differentiate resulted in reduction in MMPs,especially the secreted forms, and, in parallel, abrogation of theengraftment process.

The biomaterials of the grafts, especially the HAs, have been shown exvivo and in vivo to maintain stemness traits in cells. Since the graftsare devoid of known signals that can trigger fate determination, thefindings of donor cells that had matured into distinct adult fates,depending whether the graft was placed onto the liver or the pancreas,implicate the local microenvironment of the host tissue as the logicalsource of relevant factors for the maturational processes.

The numbers of cells that can be engrafted are considerable (>10⁸) anddictated by the dimensions of the graft, the numbers of cells, and therepertoire of secreted and plasma-membrane-associated MMPs. Thesefindings are in contrast to the limited numbers of cells (e.g. 10⁵-10⁶)feasible with vascular delivery or by injection grafting.

Patch grafting is a safe strategy by which to transplant large numbersof cells into a solid organ, including internal organs, and may proveuseful for treatment of patients especially if engraftment can occursufficiently under disease conditions. Although, there is concern thataberrant engraftment may occur where tissue is fibrotic or affected bycirrhosis. Accordingly, examples are provided herein to determineefficacy of patch grafts for the method aspects.

Example 2: Treatment of Liver Disease

This example describes an exemplary method of treating a subject havinga liver disease or disorder using a patch graft. Donor cells areprepared as organoids of biliary tree stem cells (BTSCs), precursors toliver and to pancreas, aggregated with early lineage stage mesenchymalcells (ELSMCs) consisting of angioblasts and their early lineage stagedescendants, precursors to endothelia and precursors to stellate cellsas described herein. The BTSC/ELSMCs organoids are embedded into softhyaluronan hydrogels (<200 Pa) placed onto a backing that is tethered toa target site of the subject's liver.

Following administration of the patch graft, the subject is monitoredfor improvement in liver function. Commonly used tests to check liverfunction include but are not limited to the alanine transaminase (ALT),aspartate aminotransferase (AST), alkaline phosphatase (ALP), albumin,and bilirubin tests. The ALT and AST tests measure enzymes that arereleased by the liver in response to damage or disease. The albumin andbilirubin tests measure how well the liver creates albumin, a protein,and how well it disposes of bilirubin, a waste product of the blood. Itis expected that after about 2 weeks to about 36 weeks, an improvementin liver function will be detected. Improvement is determined bydetecting an improved value of one or more of the liver function testsrelative to the value prior to administration of the graft and/or animprovement or amelioration of one or more symptoms of the liver diseaseor disorder.

Example 3: Treatment of Pancreatic Disease

This example describes an exemplary method of treating a subject havinga disease or disorder of the pancreas using a patch graft. Donor cellsare prepared as organoids of biliary tree stem cells (BTSCs), aggregatedwith early lineage stage mesenchymal cells (ELSMCs) consisting ofangioblasts and their early lineage stage descendants, precursors toendothelia and precursors to stellate cells as described herein. TheBTSC/ELSMCs organoids are embedded into soft hyaluronan hydrogels (<200Pa) placed onto a backing that is tethered to a target site of thesubject's pancreas.

Following administration of the patch graft, the subject is monitoredfor improvement in pancreatic function. Commonly used tests to checkpancreatic function include but are not limited to blood tests forlevels of the pancreatic enzymes amylase and lipase, the directpancreatic function test following administration of secretin orcholecystokinin, fecal elastase test, CT scan with contrast dye,abdominal ultrasound, endoscopic retrograde cholangiopancreatography(ERCP), endoscopic ultrasound, and magnetic resonancecholangiopancreatography. It is expected that after about 2 weeks toabout 36 weeks, an improvement in pancreatic function will be detected.Improvement is determined by detecting an improved value of one or moreof the pancreatic function tests relative to the value prior toadministration of the graft and/or an improvement or amelioration of oneor more symptoms of the disease or disorder of the pancreas.

Example 4: Treatment of Kidney Disease

This example describes an exemplary method of treating a subject havinga disease or disorder of the kidney using a patch graft. Donor cells areprepared as organoids of biliary tree stem cells (BTSCs), aggregatedwith early lineage stage mesenchymal cells (ELSMCs) consisting ofangioblasts and their early lineage stage descendants, precursors toendothelia and precursors to stellate cells as described herein. TheBTSC/ELSMCs organoids are embedded into soft hyaluronan hydrogels (<200Pa) placed onto a backing that is tethered to a target site of thesubject's kidney.

Following administration of the patch graft, the subject is monitoredfor improvement in kidney function. Commonly used tests to checkpancreatic function include but are not limited to clinically relevantendpoints of kidney function known in the art. It is expected that afterabout 2 weeks to about 36 weeks, an improvement in kidney function willbe detected. Improvement is determined by detecting an improved value ofone or more of the kidney function tests relative to the value prior toadministration of the graft and/or an improvement or amelioration of oneor more symptoms of the disease or disorder of the kidney.

Example 5: Treatment of GI Disease

This example describes an exemplary method of treating a subject havinga gastrointestinal disease or disorder using a patch graft. Donor cellsare prepared as organoids of biliary tree stem cells (BTSCs), aggregatedwith early lineage stage mesenchymal cells (ELSMCs) consisting ofangioblasts and their early lineage stage descendants, precursors toendothelia and precursors to stellate cells as described herein. TheBTSC/ELSMCs organoids are embedded into soft hyaluronan hydrogels (<200Pa) placed onto a backing that is tethered to a target site of thesubject's intestines.

Following administration of the patch graft, the subject is monitoredfor improvement in intestinal function. Commonly used tests to checkintestinal function include but are not limited clinically relevantendpoints of intestinal function known in the art. It is expected thatafter about 2 weeks to about 36 weeks, an improvement in intestinalfunction will be detected. Improvement is determined by detecting animproved value of one or more of the intestinal function tests relativeto the value prior to administration of the graft and/or an improvementor amelioration of one or more symptoms of the gastrointestinal diseaseor disorder.

What is claimed is:
 1. A method of treating a subject with agastrointestinal disease or disorder, the method comprising contactingone or more of the subject's intestines with a patch graft comprising:(a) a mixed population having two or more cell types, at least one ofwhich is at an early lineage stage that is capable of expressingmembrane-associated and/or secreted matrix metalloproteinases (MMPs),said mixed population supported in a medium in a hydrogel havingviscoelasticity sufficient to allow for migration of said mixedpopulation towards and into a target tissue; and (b) a backingcomprising a biocompatible, biodegradable material having aviscoelasticity sufficient to inhibit a migration of said mixedpopulation in a direction away from the target tissue and through saidbacking or barrier, and allowing the cells contained in the patch graftto become incorporated into the intestine, thereby restoring someintestinal function.
 2. The method of claim 1 in which thegastrointestinal disease or disorder is gastroenteritis,gastrointestinal cancer, ileitis, inflammatory bowel disease, Crohn'sdisease, ulcerative colitis, irritable bowel syndrome, peptic ulcerdisease, celiac disease, fibrosis, angiodysplasia, Hirschsprung'sdisease, pseudomembranous colitis, or gastrointestinal trauma.
 3. Themethod of claim 1 in which the patch graft is configured to sustain andmaintain said mixed population while inhibiting said at least one earlylineage stage cell type from differentiating or further maturing to alater lineage stage that is no longer capable of expressingmembrane-associated and/or secreted MMPs.
 4. The method of claim 1 inwhich said backing comprises a porous mesh infused with a hydrogel. 5.The method of claim 1 in which the patch graft further comprises: (c) ahydrogel overlaid on a serosal surface of said backing, the serosalsurface being opposite to that in contact with said mixed population. 6.The method of claim 1 in which the hydrogel of element (a) comprises oneor more hyaluronans.
 7. The method of claim 4 in which the hydrogel ofelement (b) comprises one or more hyaluronans.
 8. The method of claim 5in which the hydrogel of element (c) comprises one or more hyaluronans.9. The method of claim 1 in which said medium comprises Kubota's Mediumor other medium that supports “stemness.”
 10. The method of claim 1 inwhich said mixed population comprises mesenchymal cells and epithelialcells.
 11. The method of claim 10 in which the mesenchymal cellscomprise early lineage stage mesenchymal cells (ELSMCs).
 12. The methodof claim 11 in which said ELSMCs comprise one or more of angioblasts,precursors to endothelia, or precursors to stellate cells or mesenchymalstem cells (MSCs).
 13. The method of claim 10 in which said epithelialcells comprise epithelial stem cells.
 14. The method of claim 13 inwhich said epithelial cells comprise biliary tree stem cells (BTSCs).15. The method of claim 13 in which said epithelial cells comprisecommitted and/or mature epithelial cells.
 16. The method of claim 15 inwhich said committed and/or mature epithelial cells comprise matureparenchymal cells.
 17. The method of claim 16 in which said matureparenchymal cells comprise one or more of hepatocytes, cholangiocytes,and islet cells.
 18. The method of claim 10 in which said mesenchymalcells and epithelial cells both comprise stem cells.
 19. The method ofclaim 1 in which said mixed population comprises autologous and/orallogeneic cells.
 20. The method of claim 1 in which one or more celltype is genetically modified.
 21. The method of claim 1 in which thebacking comprises a porous mesh, scaffold, or membrane.
 22. The methodof claim 1 in which the backing comprises non-porous material.
 23. Themethod of claim 22 in which the non-porous material is selected fromsilk, amnion, placenta, omentum, a synthetic textile, a derivative ofthe foregoing, or combinations thereof.
 24. The method of claim 1 inwhich the backing has sufficient resilience to withstand mechanicalforces, is able to be tethered to a target organ or tissue, and hassufficient flexibility to be tethered to locations with curvature. 25.The method of claim 1 in which any biomaterial (other than a hydrogel)can be utilized in the patch graft so long as the biomaterial is capableof sustaining and maintaining the cell populations and has rheologicalproperties (e.g., viscoelasticity) sufficient to allow for migration ofsaid cell population within or away from the patch graft.